Lipid containing formulations

ABSTRACT

Compositions and methods useful in administering nucleic acid based therapies, for example association complexes such as liposomes and lipoplexes are described.

CLAIM OF PRIORITY

This application is a continuation of U.S. patent application Ser. No.12/056,230, filed on Mar. 26, 2008, which is a continuation ofInternational Application No. PCT/US2007/080331, filed Oct. 3, 2007,which claims priority to U.S. Provisional Application No. 60/828,022filed on Oct. 3, 2006 and claims priority to U.S. ProvisionalApplication No. 60/870,457 filed on Dec. 18, 2006, the entire contentsof each are hereby incorporated by reference.

TECHNICAL FIELD

This invention relates to compositions and methods useful inadministering nucleic acid based therapies, for example associationcomplexes such as liposomes and lipoplexes.

BACKGROUND

The opportunity to use nucleic acid based therapies holds significantpromise, providing solutions to medical problems that could not beaddressed with current, traditional medicines. The location andsequences of an increasing number of disease-related genes are beingidentified, and clinical testing of nucleic acid-based therapeutics fora variety of diseases is now underway.

One method of introducing nucleic acids into a cell is mechanically,using direct microinjection. However this method is not generallyeffective for systemic administration to a subject.

Systemic delivery of a nucleic acid therapeutic requires distributingnucleic acids to target cells and then transferring the nucleic acidacross a target cell membrane intact and in a form that can function ina therapeutic manner.

Viral vectors have, in some instances, been used clinically successfullyto administer nucleic acid based therapies. However, while viral vectorshave the inherent ability to transport nucleic acids across cellmembranes, they can pose risks. One such risk involves the randomintegration of viral genetic sequences into patient chromosomes,potentially damaging the genome and possibly inducing a malignanttransformation. Another risk is that the viral vector may revert to apathogenic genotype either through mutation or genetic exchange with awild type virus.

Lipid-based vectors have also been used in nucleic acid therapies andhave been formulated in one of two ways. In one method, the nucleic acidis introduced into preformed liposomes or lipoplexes made of mixtures ofcationic lipids and neutral lipids. The complexes thus formed haveundefined and complicated structures and the transfection efficiency isseverely reduced by the presence of serum. The second method involvesthe formation of DNA complexes with mono- or poly-cationic lipidswithout the presence of a neutral lipid. These complexes are prepared inthe presence of ethanol and are not stable in water. Additionally, thesecomplexes are adversely affected by serum (see, Behr, Acc. Chem. Res.26:274-78 (1993)).

SUMMARY

The invention features novel preparations that include a polyaminecompound or a lipid moiety described herein.

In some embodiments, the invention features a preparation comprising oneor more compounds, each individually having a structure defined byformula (I) or a pharmaceutically acceptable salt thereof,

wherein

-   -   each X^(a) and X^(b), for each occurrence, is independently C₁₋₆        alkylene;    -   n is 0, 1, 2, 3, 4, or 5; each R is independently H,

wherein at least n+2 of the R moieties in at least about 50% of themolecules of the compound of formula (I) in the preparation (e.g., atleast about 55%, at least about 60%, at least about 65%, at least about70%, at least about 75%, at least about 80%, at least about 85%, atleast about 90%, at least about 95%, at least about 98%, at least about99%, or substantially all) are not H;

-   -   m is 1, 2, 3 or 4; Y is O, NR², or S;    -   R¹ is alkyl alkenyl or alkynyl; each of which is optionally        substituted with one or more substituents; and    -   R² is H, alkyl alkenyl or alkynyl; each of which is optionally        substituted each of which is optionally substituted with one or        more substituents;

provided that, if n=0, then at least n+3 of the R moieties are not H.

In some embodiments, when R is not H, R is R_(a), for example, when R isnot H, R is R_(a) for each occurrence.

In some embodiments, when R is not H, R is R_(b), for example, when R isnot H, R is R_(b), for each occurrence.

In some embodiments, when R is not H, R is R_(c), for example, when R isnot H, R is R_(c), for each occurrence.

In some embodiments, when R is not H, R is R_(d), for example, when R isnot H, R is R_(d), for each occurrence.

In some embodiments, when R is not H, R is R_(e), for example, when R isnot H, R is R_(e), for each occurrence.

In some embodiments, n+2 of the R moieties of formula (I) are not H. Insome embodiments, n+3 of the R moieties of formula (I) are not H. Insome embodiments, n+4 of the R moieties of formula (I) are not H.

In some embodiments, n+1 of the R moieties of formula (I) are not H.

In some embodiments, n>0, and at least one R of NR of formula (I) is H.

In some embodiments, at least one R of NR₂ of formula (I) is H.

In some embodiments, at least 80% of the molecules are a singlestructural isomer. For example, n+2 of the R moieties of formula (I) arenot H, or n+3 of the R moieties of formula (I) are not H, or n+4 of theR moieties of formula (I) are not H.

In some embodiments, n is 2 or 0.

In some embodiments, X^(a) and X^(b) are C₂ alkylene.

In some embodiments, n is O and X^(b) is ethylene or propylene.

In some embodiments, n>1 and X^(a) varies with at least one occurrence.

In some embodiments, when R not H, R is

For example, Y can be O or NR². In some embodiments, m is 2. In someembodiments, Y is O or NR² and m is 2. In some embodiments, m is 1. Insome embodiments, m is 1 and Y is O or NR².

In some embodiments, R¹ for at least one occurrence is alkyl, forexample, R¹ for each occurrence is alkyl.

In some embodiments, R¹ is alkyl and R² is H, for at least oneoccurrence, e.g., for each occurrence.

In some embodiments, R¹ and R² are alkyl for at least one occurrence,e.g., for each occurrence.

In some embodiments, R¹ for at least one occurrence is alkenyl.

In some embodiments, R¹ for at least one occurrence is alkenyl.

In some embodiments, when R is not H, R is R_(a), for at least oneoccurrence, e.g., for each occurrence, and Y is O or NH. In someembodiments, Y is O. In some embodiments, Y is NH. In some embodiments,R¹ is alkyl, e.g., C₁₀₋₃₀ alkyl or C₁₂ alkyl. In some embodiments, n is2. In some embodiments, X^(a), for each occurrence is C₂ alkylene andX^(b) is C₂ alkylene. In some embodiments, m is 2.

In some embodiments, n is 2 and R, when R is not H, is R_(a), for atleast one occurrence, e.g., for each occurrence. In some embodiments, R¹is alkyl, e.g., C₁₀₋₁₈ alkyl or C₁₂ alkyl. In some embodiments, Y is Oor Y is NH. In some embodiments, X^(a), for each occurrence is C₂alkylene and X^(b) is C₂ alkylene. In some embodiments, m is 2.

In some embodiments, at least 1 R of NR is H and R, when not H is R_(a),for at least one occurrence, e.g. for each occurrence, and Y is O or NH.In some embodiments, Y is O or Y is NH. In some embodiments, R¹ isalkyl, e.g., C₁₀₋₁₈ alkyl or C₁₂ alkyl. In some embodiments, n is 2. Insome embodiments, X^(a), for each occurrence is C₂ alkylene and X^(b) isC₂ alkylene. In some embodiments, m is 2.

In some embodiments, n is 2 and at least 1 R of NR is H and when R isnot H, R is R_(a), for at least one occurrence, e.g. for eachoccurrence, and Y is O or NH. In some embodiments, R¹ is alkyl, e.g.,C₁₀₋₁₈ alkyl or C₁₂ alkyl. In some embodiments, Y is O or Y is NH. Insome embodiments, X^(a), for each occurrence is C₂ alkylene and X^(b) isC₂ alkylene. In some embodiments, m is 2.

In some embodiments, at least 1 R of NR₂ is H and R is R_(a), for atleast one occurrence, e.g. for each occurrence, and wherein Y is O orNH. In some embodiments, Y is O or Y is NH. In some embodiments, R¹ isalkyl, e.g., C₁₀₋₃₀ alkyl, C₁₀₋₁₈ alkyl or C₁₂ alkyl. In someembodiments, n is 2. In some embodiments, X^(a), for each occurrence isC₂ alkylene and X^(b) is C₂ alkylene. In some embodiments, m is 2.

In some embodiments, n is 2 and at least 1 R of NR₂ is H and R is R_(a),for at least one occurrence, e.g. for each occurrence, and wherein Y isO or NH. In some embodiments, R¹ is alkyl, e.g., C₁₀₋₁₈ alkyl or C₁₂alkyl. In some embodiments, Y is O or Y is NH. In some embodiments,X^(a), for each occurrence is C₂ alkylene and X^(b) is C₂ alkylene. Insome embodiments, m is 2.

In some embodiments, the preparation comprises one or a mixture of theformula below, wherein R is not H unless specified in the formula below.

In some embodiments, the preparation consists essentially of one or amixture of the formula below

In some embodiments, each R is

In some embodiments, each R is

In some embodiments, R¹ is C₁₀-C₁₈ alkyl (e.g., C₁₂ alkyl), or C₁₀-C₃₀)alkenyl.

In some embodiments, R is

In some embodiments, R¹ is C₁₀-C₁₈ alkyl, e.g., C₁₂ alkyl. In someembodiments, R¹ is C₁₂ alkyl and R² is H.

In some embodiments, n is 0 and X is propylene. In some embodiments, 1 Ris H. In some embodiments, when R is not H, R is R_(a), for at least oneoccurrence, e.g. for each occurrence. In some embodiments, R¹ is alkyl,e.g., C₁₀₋₃₀ alkyl or C₁₂ alkyl. In some embodiments, Y is O or Y is NH.In some embodiments, m is 2.

In some embodiments, formula (I) is

In some embodiments, R is

In some embodiments, R¹ is C₁₀-C₁₈ alkyl, or C₁₀-C₃₀ alkenyl. In someembodiments, R is

In some embodiments, R¹ is C₁₀-C₁₈ alkyl, or C₁₀-C₃₀ alkenyl and R² isH.

In some embodiments,

-   -   n is 2;    -   X^(a), for each occurrence is C₂ alkylene and X^(b) is C₂        alkylene; and    -   wherein    -   each R is H or

-   -   -   R_(a), for at least one occurrence, e.g. for each            occurrence,

    -   m is 2;

    -   Y is NH or O;

R¹ is C₁₂ alkyl. In some embodiments, at least 80% of the molecules ofthe compound of formula (I) are a single structural isomer. In someembodiments, Y is NH, e.g., wherein at least 80% of the molecules of thecompound of formula (I) are a single structural isomer. In someembodiments, R is R^(a), for 5 occurrences. In some embodiments, in atleast 80% of the molecules of the compound of formula (I), R is R^(a),for 5 occurrences. In some embodiments, Y is NH.

In some embodiments, the compound of formula (I) is an inorganic ororganic salt thereof, e.g., a hydrohalide salt thereof, such as ahydrochloride salt thereof. In some embodiments, the hydrochloride saltranges from a single equivalent of HCL, to n+2 equivalents of HCl. Insome embodiments, the compound of formula (I) is salt of an organicacid, e.g., an acetate, for example, the acetate salt ranges from asingle equivalent of acetate, to n+2 equivalents of acetate or aformate, for example, the formate salt ranges from a single equivalentof acetate, to n+2 equivalents of formate.

In some embodiments, the compound of formula (I) is in the form of ahydrate.

In some embodiments, R¹, for at least one occurrence, e.g., for eachoccurrence, comprises an alkenyl moiety, for example, R¹ comprises a cisdouble bond.

In one aspect, the invention features a preparation including a compoundof formula (I) and a nucleic acid (e.g., an RNA such as an siRNA ordsRNA or a DNA). In some embodiment, the preparation also includes anadditional lipid such as a fusogenic lipid, or a PEG-lipid.

In some embodiments, the preparation comprises less than 11%, by weight,of

wherein X and n are defined as in formula (I) above.

In some embodiments, the preparation comprises less than 90% by weightof

-   -   wherein Y and R¹ are defined as in formula (I) above.

In some embodiments, the preparation comprises a plurality of compoundsof formula (I).

In some embodiments, the preparation comprises a mixture of compounds ofthe formulas below:

wherein in formula (I″), five of the R moieties are R^(a). In someembodiments, formula (I′) and (I″) are present in a ratio of from about1:2 to about 2:1.

In one aspect, the invention features a method of making a compound offormula

wherein

-   -   each X^(a) and X^(b), for each occurrence, is independently C₁₋₆        alkylene;    -   n is 0, 1, 2, 3, 4, or 5; and    -   wherein    -   each R is independently H or

-   -   -   R_(a);

    -   m is 2;

    -   Y is O, NR², or S;

    -   R¹ is alkyl or alkenyl;

    -   R² is H or C alkyl or alkenyl;

    -   the method comprising reacting a compound of formula (III)

-   -   with a compound of formula (IV),

-   -   in the presence of a promoter.

In one aspect, the invention features a method of making a compound offormula (II),

wherein

-   -   each X^(a) and X^(b), for each occurrence, is independently C₁₋₆        alkylene;    -   n is 0, 1, 2, 3, 4, or 5; and    -   wherein    -   each R is independently H or

-   -   -   R_(a)

    -   m is 2;

    -   Y is O, NR², or S;

    -   R¹ is alkyl or alkenyl;

    -   R² is H or C alkyl or alkenyl;

    -   the method comprising reacting a compound of formula (III)

-   -   with a compound of formula (IV),

-   -   in the presence of a quencher.

In one aspect, the invention features a method of making a compound offormula (II),

wherein

-   -   each X^(a) and X^(b), for each occurrence, is independently C₁₋₆        alkylene;    -   n is 0, 1, 2, 3, 4, or 5; and    -   wherein    -   each R is independently H or

-   -   -   R_(a);

    -   m is 2;

    -   Y is O, NR², or S;

    -   R¹ is alkyl or alkenyl;

    -   R² is H or alkyl or alkenyl; the method comprising reacting a        compound of formula (III)

-   -   with a compound of formula (IV),

-   -   wherein the reaction mixture comprises from about 0.8 about 1.2        molar equivalents of a compound of formula (III), with from        about 3.8 to about 6.5 molar equivalents of a compound of        formula (IV).

In some embodiments, the reaction mixture comprises from about 0.8 about1.2 molar equivalents of a compound of formula (III), with from about5.5 to about 6.5 molar equivalents of a compound of formula (IV). Insome embodiments, the reaction mixture comprises about 1 molarequivalents of a compound of formula (III), with from about 6 molarequivalents of a compound of formula (IV). In some embodiments, thereaction mixture comprises about 1 molar equivalents of a compound offormula (III), with from about 5 molar equivalents of a compound offormula (IV).

In one aspect, the invention features a method of making a compound offormula (II),

wherein

-   -   each X^(a) and X^(b), for each occurrence, is independently C₁₋₆        alkylene;    -   n is 0, 1, 2, 3, 4, or 5; and    -   wherein    -   each R is independently H or

-   -   -   R_(a);

    -   m is 2;

    -   Y is O, NR², or S;

    -   R¹ is alkyl or alkenyl;

    -   R² is H or alkyl or alkenyl;

    -   the method comprising a two step process of reacting a compound        of formula (III)

-   -   with a compound of formula (IV),

-   -   in the presence of boric acid and water    -   wherein, the first step process involving the reaction mixture        comprises from about 0.8 about 1.2 molar equivalents of a        compound of formula (III), with from about 3.8 to about 4.2        molar equivalents of a compound of formula (IV) and the second        step process involving addition of about 0.8 to 1.2 molar        equivalent of compound of formula (IV).

In one aspect, the invention features a method of making a compound offormula (II),

wherein

-   -   each X^(a) and X^(b), for each occurrence, is independently C₁₋₆        alkylene;    -   n is 0, 1, 2, 3, 4, or 5; and    -   wherein    -   each R is independently H or

-   -   -   R_(a)

    -   m is 2;

    -   Y is O, NR², or S;

    -   R¹ is alkyl or alkenyl;

    -   R² is H or alkyl or alkenyl;

    -   the method comprising reacting a compound of formula (III)

-   -   with a compound of formula (IV),

-   -   and separating at least one structural isomer of formula (II)        from the reaction mixture to provide a substantially purified        preparation comprising a structural isomer of formula (II).

In some embodiments, the structural isomer of formula (II) is separatedfrom the reaction mixture using chromatographic separation. In someembodiments, the chromatographic separation is using flash silica gelfor separation of isomers. In some embodiments, the chromatographicseparation is gravity separation of isomers using silica gel. In someembodiments, the chromatographic separation is using moving bedchromatography for separation of isomers. In some embodiments, thechromatographic separation uses liquid chromatography (LC) forseparation of isomers. In some embodiments, the chromatographicseparation is normal phase HPLC for separation of isomers. In someembodiments, the chromatographic separation is reverse phase HPLC forseparation of isomers.

In some embodiments, the substantially purified preparation comprises atleast about 80% of the structural isomer of formula (II), e.g., at leastabout 90% of the structural isomer of formula (II), at least about 95%of the structural isomer of formula (II).

In another aspect, the invention features a method of making a compoundof formula (V) or a pharmaceutically acceptable salt thereof,

wherein

-   -   each X^(a) and X^(b), for each occurrence, is independently C₁₋₆        alkylene;    -   n is 0, 1, 2, 3, 4, or 5; and    -   wherein    -   each R is independently H or

-   -   -   R_(a);

    -   m is 1;

    -   Y is O, NR², or S;

    -   R¹ is alkyl or alkenyl;

    -   R² is H or alkyl or alkenyl;

    -   the method comprising reacting a compound of formula (III)

-   -   with a compound of formula (VI),

-   -   to provide a compound of formula (V) or a pharmaceutically        acceptable salt thereof.

In some embodiments, the pharmaceutically acceptable salt thereof is ahydrochloride salt of the compound of formula (V).

In one aspect, the invention features a compound of formula (X),

wherein

R¹ and R² are each independently H, C₁-C₆ alkyl, optionally substitutedwith 1-4 R⁵, C₂-C₆ alkenyl, optionally substituted with 1-4 R⁵, orC(NR⁶)(NR⁶)₂;

R³ and R⁴ are each independently alkyl, alkenyl, alkynly, each of whichis optionally substituted with fluoro, chloro, bromo, or iodo;

L¹ and L² are each independently —NR⁶C(O)—, —C(O)NR⁶—, —OC(O)—, —C(O)O—,—S—S—, —N(R⁶)C(O)N(R⁶)—, —OC(O)N(R⁶)—, —N(R⁶)C(O)O—, —O—N═C—, OR,—OC(O)NH—N═C—, or —NHC(O)NH—N═C—,

L¹-R³ and L²-R⁴ can be taken together to form an acetal, a ketal, or anorthoester, wherein R³ and R⁴ are defined as above and can also be H orphenyl;

R⁵ is fluoro, chloro, bromo, iodo, —OR⁷, —N(R⁸)(R⁹), —CN, SR¹⁰, S(O)R¹⁰,S(O)₂R¹⁰

R⁶ is H, C₁-C₆ alkyl,

R⁷ is H or C₁-C₆ alkyl;

each R⁸ and R⁹ are independently H or C₁-C₆ alkyl;

R¹⁰ is H or C₁-C₆ alkyl;

m is 1, 2, 3, 4, 5, or 6;

n is 0, 1, 2, 3, 4, 5, or 6;

and pharmaceutically acceptable salts thereof.

In some embodiments, the compound is an inorganic salt thereof, forexample a hydrohalide salt thereof such as a hydrochloride salt thereof.In some embodiments, the compound is an organic salt thereof.

In some embodiments, R¹ and R² are each independently C₁-C₃ alkyl.

In some embodiments, R¹ is methyl.

In some embodiments, R² is methyl.

In some embodiments, R¹ and R² are both methyl.

In some embodiments, R¹ is H, methyl, ethyl, isopropyl, or2-hydroxyethyl.

In some embodiments, R² is H.

In some embodiments, R² is methyl, ethyl, propyl, or isopropyl.

In some embodiments, R¹ is H, methyl, ethyl, isopropyl, or2-hydroxyethyl and R² is H, methyl, ethyl, propyl, or isopropyl.

In some embodiments, m is 1.

In some embodiments, n is 1.

In some embodiments, both m and n are 1.

In some embodiments, L¹ is —NR⁶C(O)—, or —C(O)NR⁶—.

In some embodiments, L¹ is −OC(O)— or —C(O)O—.

In some embodiments, L¹ is S—S—.

In some embodiments, L¹ is —N(R⁶)C(O)N(R⁶)—.

In some embodiments, L¹ is —OC(O)N(R⁶)— or —N(R⁶)C(O)O—.

In some embodiments, L¹ is —O—N═C—.

In some embodiments, L¹-OC(O)NH—N═C— or —NHC(O)NH—N═C—. In someembodiments, L² is —NR⁶C(O)—, or —C(O)NR⁶—.

In some embodiments, L² is −OC(O)— or —C(O)O—.

In some embodiments, L² is S—S—.

In some embodiments, L² is —N(R⁶)C(O)N(R⁶)—.

In some embodiments, L² is —OC(O)N(R⁶)— or —N(R⁶)C(O)O—.

In some embodiments, L² is —O—N═C—.

In some embodiments, L²-OC(O)NH—N═C— or —NHC(O)NH—N═C—.

In some embodiments, both L¹ and L² are —NR⁶C(O)—, or —C(O)NR⁶—.

In some embodiments, both L¹ and L² are —OC(O)— or —C(O)O—.

In some embodiments, both L¹ and L² are S—S—.

In some embodiments, both L¹ and L² are —N(R⁶)C(O)N(R⁶)—.

In some embodiments, both L¹ and L² are —OC(O)N(R⁶)— or —N(R⁶)C(O)O—.

In some embodiments, L¹ is —NR⁶C(O)— and L² is —S—S—.

In some embodiments, L¹ is —OC(O)— and L² is —S—S—.

In some embodiments, L¹ is —OC(O)N(R⁶) or —N(R⁶)C(O)O— and L² is —S—S—.

In some embodiments, L¹ is —N(R⁶)C(O)N(R⁶)— and L² is —S—S—.

In some embodiments, L¹-R³ and L²-R⁴ are taken together to form anacetal, a ketal, or an orthoester.

In some embodiments, each R³ and R⁴ are independently alkyl.

In some embodiments, both R³ and R⁴ are C₆-C₂₈ alkyl.

In some embodiments, each L¹ and L² are independently —S—S—,—OC(O)N(R⁶)— or —N(R⁶)C(O)O—.

In some embodiments, R³ is alkyl.

In some embodiments, R⁴ is alkyl.

In some embodiments, R³ is alkenyl.

In some embodiments, R⁴ is alkenyl.

In some embodiments, each R³ and R⁴ are independently alkenyl, forexample, each R³ and R⁴ are independently C₆-C₃₀ alkenyl or each R³ andR⁴ are the same alkenyl moiety.

In some embodiments, each R³ and R⁴ includes two double bond moieties.In some embodiments, at least one of the double bonds have a Zconfiguration. In some embodiments, both of the double bonds have a Zconfiguration. In some embodiments, at least one of R³ and R⁴ isprovided in formula (II) below

wherein

x is an integer from 1 to 8; and

y is an integer from 1-10. In some embodiments, both of R³ and R⁴ are ofthe formula (II). In some embodiments, at least one of the double bondshave an E configuration, e.g., both of the double bonds have an Econfiguration. In some embodiments, at least one of R¹ and R² isprovided in formula (III) below

-   -   wherein    -   x is an integer from 1 to 8; and    -   y is an integer from 1-10.

In some embodiments, each R¹ and R² includes three double bond moieties.In some embodiments, at least one of the double bonds have a Zconfiguration. In some embodiments, at least two of the double bondshave a Z configuration. In some embodiments, all three of the doublebonds have a Z configuration. In some embodiments, at least one of R¹and R² is provided in formula (IV) below

-   -   wherein    -   x is an integer from 1 to 8; and    -   y is an integer from 1-10. In some embodiments, both of R¹ and        R² are as provided in formula (IV). In some embodiments, at        least one of the double bonds have an E configuration. In some        embodiments, at least two of the double bonds have an E        configuration. In some embodiments, all three of the double        bonds have an E configuration. In some embodiments, at least one        of R¹ and R² is provided in formula (IV) below

-   -   wherein    -   x is an integer from 1 to 8; and    -   y is an integer from 1-10. In some embodiments, both of R¹ and        R² are as provided in formula (V).

In some embodiments, R¹ and R² are each C₁-C₆ alkyl (e.g., methyl), L1and L1 are each —OC(O)—, and R³ and R⁴ are each alkenyl. In someembodiments, R³ and R⁴ are the same. In some embodiments, R³ and R⁴ bothinclude two double bonds (e.g., having cis linkages). In someembodiments R³ and R⁴ are provided in formula (II) below

wherein

x is an integer from 1 to 8 e.g., 5; and

y is an integer from 1-10 e.g., 4.

In one aspect, the invention features a preparation including a compoundof formula (X).

In one aspect, the invention features a preparation including a compoundof formula (X) and a nucleic acid (e.g., an RNA such as an siRNA ordsRNA or a DNA). In some embodiment, the preparation also includes anadditional lipid such as a fusogenic lipid, or a PEG-lipid.

In one aspect, the invention features a method of making a compound offormula (X),

wherein

R¹ and R² are each independently C₁-C₆ alkyl, optionally substitutedwith 1-4 R⁵;

R³ is alkyl, alkenyl, alkynyl

L¹ is —OC(O)—

R⁵ is −OR⁷, —N(R⁸)(R⁹), —CN, SR¹⁰, S(O)R¹⁰, S(O)₂R¹⁰

R⁶ is H, C₁-C₆ alkyl;

R⁷ is H or C₁-C₆ alkyl;

each R⁸ and R⁹ are independently H or C₁-C₆ alkyl;

R¹⁰ is H or C₁-C₆ alkyl;

m and n are each independently 1, 2, 3, 4, 5, or 6,

the method comprising reacting a compound of formula (VI),

with a compound of formula (VII)

in the presence of a coupling agent, thereby providing a compound offormula (X).

In some embodiments, the coupling agent is a carbodiimide such as EDCI.

In one aspect, the invention features a compound of formula (XV) below

wherein;

each L¹ and L² are independently a bond or C(O);

each R¹ and R² are independently alkyl alkenyl or alkynyl; each of whichis optionally substituted with one or more substituents;

X is —C(O)NH—, C(S)NH, —C(O)C₁₋₃alkylC(O)NH—; or —C(O)C₁₋₃alkylC(O)O—;

m is an integer from 0-11 and

n is an integer from 1-500.

In some embodiments, L¹ and L² are both a bond.

In some embodiments, L¹ and L² are both C(O).

In some embodiments, each R¹ and R² are independently alkyl, for exampleC₆-C₂₈ alkyl, e.g., C₁₀-C₁₈ alkyl, e.g., C₁₃ alkyl, C₁₄ alkyl, C₁₅alkyl, or C₁₆ alkyl, In some embodiments, both R¹ and R² are alkyl,e.g., straight chain alkyl having the same length, e.g., C₆-C₂₈ alkyl,e.g., C₁₀-C₁₈ alkyl, e.g., C₁₃ alkyl, C₁₄ alkyl, C₁₅ alkyl, or C₁₆alkyl. In some preferred embodiments, both R¹ and R² are C₁₄ alkyl.

In some embodiments, the formula XV represents a racemic mixture

In some embodiments, the compound of formula XV has an enantiomericexcess of the R isomer, e.g., at least about 65%, 70%, 75%, 80%, 85%,90%, 95%, 97%, 98%, or 99%. In some embodiments the formula XVrepresents enantiomerically pure ‘R’ isomer.

In some embodiments, the compound of formula XV has an enantiomericexcess of the S isomer, e.g., at least about 65%, 70%, 75%, 80%, 85%,90%, 95%, 97%, 98%, or 99%. In some embodiments the formula XVrepresents enantiomerically pure ‘S’ isomer.

In some embodiments, each R¹ and R² are independently alkenyl, forexample, each R¹ and R² are independently C₆-C₃₀ alkenyl or each R¹ andR² are the same alkenyl moiety. In some embodiments, each R¹ and R²includes a single double bond, for example a single double bond in the Eor Z configuration.

In some embodiments, each R¹ and R² includes two double bond moieties.In some embodiments, at least one of the double bonds has a Zconfiguration. In some embodiments, both of the double bonds have a Zconfiguration. In some embodiments, at least one of R¹ and R² isprovided in formula (II) below

wherein

x is an integer from 1 to 8; and

y is an integer from 1-10. In some embodiments, both of R¹ and R² are ofthe formula (II). In some embodiments, at least one of the double bondshas an E configuration, e.g., both of the double bonds have an Econfiguration. In some embodiments, at least one of R¹ and R² isprovided in formula (III) below

wherein

x is an integer from 1 to 8; and

y is an integer from 1-10.

In some embodiments, each R¹ and R² includes three double bond moieties.In some embodiments, at least one of the double bonds has a Zconfiguration. In some embodiments, at least two of the double bondshave a Z configuration. In some embodiments, all three of the doublebonds have a Z configuration. In some embodiments, at least one of R¹and R² is provided in formula (IV) below

wherein

x is an integer from 1 to 8; and

y is an integer from 1-10. In some embodiments, both of R¹ and R² are asprovided in formula (IV). In some embodiments, at least one of thedouble bonds has an E configuration. In some embodiments, at least twoof the double bonds have an E configuration. In some embodiments, allthree of the double bonds have an E configuration. In some embodiments,at least one of R¹ and R² is provided in formula (IV) below

wherein

x is an integer from 1 to 8; and

y is an integer from 1-10. In some embodiments, both of R³ and R⁴ are asprovided in formula (V).

In some embodiments, X is —C(O)NH—, providing a compound of formula(XV') below:

In some embodiments, each R¹ and R² are independently alkyl, for exampleC₆-C₂₈ alkyl, e.g., C₁₀-C₁₈ alkyl, e.g., C₁₃ alkyl, C₁₄ alkyl, C₁₅alkyl, or C₁₆ alkyl. In some embodiments, both R¹ and R² are alkyl,e.g., straight chain alkyl having the same length, e.g., C₆-C₂₈ alkyl,e.g., C₁₀-C₁₈ alkyl, e.g., C₁₃ alkyl, C₁₄ alkyl, C₁₅ alkyl, or C₁₆alkyl. In some preferred embodiments, both R¹ and R² are C₁₄ alkyl.

In some embodiments, X is —C(O)C₁₋₃alkylC(O)O—.

In some embodiments, m is an integer from 1-10, for example an integerfrom 2-4 or an integer 2.

In some embodiments, n is an integer from 1-500, for example an integerfrom 40-400, from 100-350, from 40-50 or from 42-47.

In some embodiments, the compound is a compound of formula (XV′),

wherein both L¹ and L² are a bond. In some embodiments, each R¹ and R²are independently alkyl, for example C₆-C₂₈ alkyl, e.g., C₁₀-C₁₈ alkyl,e.g., C₁₄ alkyl, C₁₅ alkyl, or C₁₆ alkyl. In some embodiments, both R¹and R² are alkyl, e.g., straight chain alkyl having the same length,e.g., C₆-C₂₈ alkyl, e.g., C₁₀-C₁₈ alkyl, e.g., C₁₄ alkyl, C₁₅ alkyl, orC₁₆ alkyl. In some preferred embodiments, both R¹ and R² are C₁₄ alkyl.In some embodiments, m is an integer from 1-10, for example an integerfrom 2-4 or an integer 2 In some embodiments, n is an integer from1-500, for example an integer from 40-400, or from 40-50.

In some embodiments, the compound is a compound of formula (XV'),wherein L1 and L2 are both bonds, R¹ and R² are both alkyl (e.g., C₆-C₂₈alkyl, e.g., C₁₀-C₁₈ alkyl, preferrably C₁₄ alkyl), and n is an integerfrom about 40-400.

In some embodiments, the compound has a formula (XVI) below:

wherein the repeating PEG moiety has an average molecular weight of 2000with n value between 42 and 47.

In some embodiments, the compound of formula XV has an enantiomericexcess of the R isomer, e.g., at least about 65%, 70%, 75%, 80%, 85%,90%, 95%, 97%, 98%, or 99%. In some embodiments the compound of formulaXVI is a stereo isomer with preferred absolute configuration ‘R’.

In one aspect, the invention features a PEG lipid conjugated to acholesterol moiety. For example, the compound of formula (XX) below:

X is —C(O)NH—, C(S)NH, —C(O)C₁₋₃alkylC(O)NH—; or —C(O)C₁₋₃alkylC(O)O—;

M is an integer from 0-11 and

n is an integer from 1-500.

In some embodiments the 0 attached to the cholesterol in formula (XX) ispart of the cholesterol moiety.

In some preferred embodiments, X is —C(O)NH—, or —C(O)C₁₋₃alkylC(O)O—.

In some embodiments, the compound of formula (XX) is as provided belowin formula (XX′)

In one aspect, the invention features a PEG lipid bound to a targetingmoiety, for example a sugar residue. For example, the compounds offormula (XV) or (XX) are modified at the OMe terminal end with atargeting moiety. In some embodiments, the targeting moiety is bound tothe PEG moiety via a linker Examplary targeted PEG lipids are providedin formulas (XXI) and (XXII) below.

In one embodiment, the lipid is a compound of formula (XXI)

wherein;

each L¹ and L² are independently a bond or C(O);

each R¹ and R² are independently alkyl alkenyl or alkynyl; each of whichis optionally substituted with one or more substituents;

each X and X′ is independently —C(O)NH—, —NHC(O)—, C(S)NH, C(S)NH,—C(O)C₁₋₃alkylC(O)NH—; NHC(O)C₁₋₃alkylC(O)—; —C(O)C₁₋₃alkylC(O)O—;NHC(O)C₁₋₃alkyl-; or C₁₋₃alkylC(O)NH—;

m is an integer from 0-11 and

n is an integer from 1-500

p is an integer from 1-6, e.g., 3;

T is a targeting moiety such as a glycosyl moiety (e.g., a sugarresidue). Examplary targeting moieties include

In some embodiments, L¹ and L² are both a bond.

In some embodiments, L¹ and L² are both C(O).

In some embodiments, each R¹ and R² are independently alkyl, for exampleC₆-C₂₈ alkyl, e.g., C₁₀-C₁₈ alkyl, e.g., C₁₄ alkyl, C₁₅ alkyl, or C₁₆alkyl. In some embodiments, both R¹ and R² are alkyl, e.g., straightchain alkyl having the same length, e.g., C₆-C₂₈ alkyl, e.g., C₁₀-C₁₈alkyl, e.g., C₁₄ alkyl, C₁₅ alkyl, or C₁₆ alkyl. In some preferredembodiments, both R¹ and R² are C₁₄ alkyl.

In some embodiments, the formula (XXI) represents a racemic mixture

In some embodiments, the compound of formula (XXI) has an enantiomericexcess of the R isomer, e.g., at least about 65%, 70%, 75%, 80%, 85%,90%, 95%, 97%, 98%, or 99%. In some embodiments the formula (XXI)represents enantiomerically pure ‘R’ isomer.

In some embodiments, the compound of formula (XXI) has an enantiomericexcess of the S isomer, e.g., at least about 65%, 70%, 75%, 80%, 85%,90%, 95%, 97%, 98%, or 99%. In some embodiments the formula (XXI)represents enantiomerically pure ‘S’ isomer.

In some embodiments, each R¹ and R² are independently alkenyl, forexample, each R¹ and R² are independently C₆-C₃₀ alkenyl or each R¹ andR² are the same alkenyl moiety. In some embodiments, each R¹ and R²includes a single double bond, for example a single double bond in the Eor Z configuration.

In some embodiments, each R¹ and R² includes two double bond moieties.In some embodiments, at least one of the double bonds has a Zconfiguration. In some embodiments, both of the double bonds have a Zconfiguration. In some embodiments, at least one of R¹ and R² isprovided in formula (II) below

wherein

x is an integer from 1 to 8; and

y is an integer from 1-10. In some embodiments, both of R¹ and R² are ofthe formula (II). In some embodiments, at least one of the double bondshas an E configuration, e.g., both of the double bonds have an Econfiguration. In some embodiments, at least one of R¹ and R² isprovided in formula (III) below

wherein

x is an integer from 1 to 8; and

y is an integer from 1-10.

In some embodiments, each R¹ and R² includes three double bond moieties.In some embodiments, at least one of the double bonds has a Zconfiguration. In some embodiments, at least two of the double bondshave a Z configuration. In some embodiments, all three of the doublebonds have a Z configuration. In some embodiments, at least one of R¹and R² is provided in formula (IV) below

wherein

x is an integer from 1 to 8; and

y is an integer from 1-10. In some embodiments, both of R¹ and R² are asprovided in formula (IV). In some embodiments, at least one of thedouble bonds has an E configuration. In some embodiments, at least twoof the double bonds have an E configuration. In some embodiments, allthree of the double bonds have an E configuration. In some embodiments,at least one of R¹ and R² is provided in formula (IV) below

wherein

x is an integer from 1 to 8; and

y is an integer from 1-10. In some embodiments, both of R³ and R⁴ are asprovided in formula (V).

In some embodiments, p is 3.

In some embodiments, L is NHC(O)C₁₋₆ alkyl (e.g., NHC(O)C₃alkyl). Insome embodiments, the compound of formula (XXI) is the compound of(XXI′) below:

In one embodiment, the lipid is a compound of formula (XXII)

wherein;

each X and X′ is independently —C(O)NH—, —NHC(O)—, C(S)NH, C(S)NH,—C(O)C₁₋₃alkylC(O)NH—; NHC(O)C₁₋₃alkylC(O)—; —C(O)C₁₋₃alkylC(O)O—;NHC(O)C₁₋₃alkyl-; or C₁₋₃alkylC(O)NH—;

m is an integer from 0-11 and

n is an integer from 1-500

p is an integer from 1-6, e.g., 3;

T is a targeting moiety such as a glycosyl moiety (e.g., a sugarresidue). Examplary targeting moieties include

In some preferred embodiments, the compound of formula (XXII) is thecompound of (XXII′) as provided below:

In one aspect, the invention features an association complex comprisinga compound preparation comprising a compound described herein (e.g., acompound of formula (I) or a compound of formula (X)) and a nucleic acidsuch as an RNA a single stranded or double stranded RNA (e.g., siRNA ordsRNA or a DNA). In some embodiments, the association complex is alipoplex or a liposome. In some embodiments the association complexincludes one or more additional components such as a targeting moiety, afusogenic lipid, a PEGylated lipid, such as a PEG-lipid described hereinsuch as a PEG-lipid having the formula (XV), (XV') or (XVI) or astructural component. In some embodiments, the PEG-lipid is a targetedPEG-lipid as described herein, e.g., a compound of formula (XXI),(XXI′), (XXII), or (XXII′).

In one aspect, the invention features a method of forming a liposomecomprising contacting a lipid preparation comprising a compounddescribed herein (e.g. a lipid described herein such as a compound offormula (I) or formula (X)) with a therapeutic agent in the presence ofa buffer, wherein said buffer:

-   -   is of sufficient strength that substantially all amines of the        molecules formula I are protonated;    -   is present at between 100 and 300 mM;    -   is present at a concentration that provides significantly more        protonation of than does the same buffer at 20 mM.

In one aspect, the invention features a liposome made by the methoddescribed herein.

In one aspect, the invention features a method of forming a liposomecomprising contacting a lipid preparation described herein (e.g., alipid preparation comprising a compound of formula (I) or a compound offormula (X)) with a therapeutic agent in a mixture comprising at leastabout 90% ethanol and rapidly mixing the lipid preparation with thetherapeutic agent to provide a particle having a diameter of less thanabout 200 uM. In some embodiments, the particle has a diameter of lessthan about 50 uM.

In one aspect, the invention features a method of forming a liposomecomprising contacting a lipid preparation described herein (e.g., alipid preparation comprising a compound of formula (I) or a compound offormula (X)) with a therapeutic agent in the presence of a buffer,wherein said buffer has a concentration from about 100 to about 300 mM.

In one aspect, the invention features liposome comprising a preparationdescribed herein (e.g., a lipid preparation comprising a compound offormula (I) or a compound of formula (X)) and a nucleic acid. In someembodiments, the preparation also includes a PEGylated lipid, forexample a PEG-lipid described herein, such as a PEG-lipid having theformula (XV), (XV') or (XVI). In some embodiments, the PEG-lipid is atargeted PEG-lipid as described herein, e.g., a compound of formula(XXI), (XXI′), (XXII), or (XXII′). In some embodiments, the preparationalso includes a structural moiety such as cholesterol. In someembodiments the preparation of association complex includes compounds offormaulae (I), (XV) and cholesterol. In some embodiments, said nucleicacid is an siRNA, for example said nucleic acid is an siRNA which hasbeen modified to resist degradation, said nucleic acid is an siRNA whichhas been modified by modification of the polysaccharide backbone, orsaid siRNA targets the ApoB gene.

In some embodiments, the liposome further comprises a structural moietyand a PEGylated lipid, such as a PEG-lipid described herein, wherein theratio, by weight, of preparation (e.g., a lipid preparation comprising acompound of formula (I) or a compound of formula (X)), a structuralmoiety such as cholesterol, PEGylated lipid, and a nucleic acid, is8-22:4-10:4-12:0.4-2.2. In some embodiments, the structural moiety ischolesterol. In some embodiments, the ratio is10-20:0.5-8.0:5-10:0.5-2.0, e.g., 15:0.8:7:1. In some embodiments, theaverage liposome diameter is between 10 nm and 750 nm, e.g., the averageliposome diameter is between 30 and 200 nm or the average liposomediameter is between 50 and 100 nm. In some embodiments, the preparationis less than 15%, by weight, of unreacted lipid. In some embodiments,the ratio of the preparation (e.g., a lipid preparation comprising acompound of formula (I) or a compound of formula (X)), the structuralmoiety such as cholesterol, and the PEG lipid is about 42/48/10 (molarratio). In some embodiments, the total lipid to nucleic acid (e.g.,siRNA) is about 7.5% by weight.

In some embodiments an association complex described herein has a weightratio of total excipients to nucleic acid of less than about 15:1, forexample, about 10:1, 7.5:1 or about 5:1.

In one aspect, the invention features a method of forming an associationcomplex comprising a plurality of lipid moieties and a therapeuticagent, the method comprising: mixing a plurality of lipid moieties inethanol and aqueous NaOAc buffer to provide a particle; and adding thetherapeutic agent to the particle, thereby forming the associationcomplex.

In some embodiments, the lipid moieties are provided in a solution of100% ethanol.

In some embodiments, the plurality of lipid moieties comprise a cationiclipid.

In some embodiments, the cationic lipid is a lipid described herein, forexample, the cationic lipid is a lipid of one of the following or amixture thereof:

In some preferred embodiments, the cationic lipid is

In some embodiments, the plurality of lipid moieties comprise aPEG-lipid, for example, the PEG-lipid has the following structure:

-   wherein;-   each L¹ and L² are independently a bond or C(O);-   each R¹ and R² are independently alkyl alkenyl or alkynyl; each of    which is optionally substituted with one or more substituents;-   X is —C(O)NH—, C(S)NH, —C(O)C₁₋₃alkylC(O)NH—; or    —C(O)C₁₋₃alkylC(O)O—;-   m is an integer from 0-11 and-   n is an integer from 1-500.

In some preferred embodiments, the PEG-lipid is a PEG lipid of formula(XVI), wherein the repeating PEG moiety has an average molecular weightof 2000, for example, with an n value between 42 and 47 or the lipidprovided below:

In some embodiments, the plurality of lipid moieties comprises astructural lipid, for example, the structural lipid is cholesterol.

In some embodiments, the PEG-lipid is a targeted PEG-lipid as describedherein, e.g., a compound of formula (XXI), (XXI′), (XXII), or (XXII′).

In some embodiments, the method includes further comprising extrudingthe lipid containing particles, for example, prior to addition of thetherapeutic agent.

In some embodiments, the therapeutic agent is a nucleic acid, forexample, an siRNA, such as an siRNA which has been modified to resistdegradation, an siRNA which has been modified by modification of thepolysaccharide backbone, or an siRNA conjugated to a Lipophilic moiety.In some embodiments, the siRNA targets the ApoB gene.

In some embodiments, the association complex comprises a cationic lipid,a structural lipid, a PEG-lipid and a nucleic acid. In some embodiments,the molar ratio of the cationic lipid, structural lipid, PEG-lipid andnucleic acid is 36-48:42-54:6-14, for example, 38-46:44-52:8-12 or about42:48:10. In some embodiments, the weight ratio of total excipient tonucleic acid is less than about 15:1, for example, about 10:1 about7.5:1 or about 5:1. In some preferred embodiments, the cationic lipidhas the following structure;

the PEG-lipid is a PEG lipid of formula (XVI), wherein the repeating PEGmoiety has an average molecular weight of 2000, for example, with an nvalue between 42 and 47 or has the following structure:

and the structural lipid is cholesterol, for example, wherein the molarratio of the cationic lipid, structural lipid, is PEG-lipid is38-46:44-52:8-12, e.g., about 42:48:10. In some preferred embodiments,the weight ratio of total exipient to nucleic acid is less than about15:1, e.g., about 10:1, about 7.5:1, or about 5:1.

In another aspect, the invention features an association complex madefrom a method described herein.

In another aspect, the invention features association complex comprisinga cationic lipid, a structural lipid, a PEG-lipid and a nucleic acid,wherein the cationic lipid is a lipid of one of the following or amixture thereof:

the PEG-lipid is a PEG lipid of formula (XVI), wherein the repeating PEGmoiety has an average molecular weight of 2000, for example, with an nvalue between 42 and 47 or has the following structure:

and the structural lipid is cholesterol. In some preferred embodiments,the nucleic acid is an siRNA. In some preferred embodiments, thecationic lipid has the following formula:

In some preferred embodiments, the molar ratio of the cationic lipidpreparation, structural lipid (e.g., cholesterol), PEG-lipid and nucleicacid is 36-48:42-54:6-14, for example, 38-46:44-52:8-12 or about42:48:10. In some preferred embodiments, the weight ratio of totalexipient to nucleic acid is less than about 15:1, for example, about10:1, about 7.5:1, or about 5:1.

In some embodiments, an association complex described herein has a meandiameter or particle size of less than about 25000 nm, e.g., from about20 to 200 nm, about 60, or about 50 nm.

In some embodiments, a nucleic acid as administered in an associationcomplex described herein, demonstrates a serum half life (e.g., invitro) for at least about 4 hours, e.g., at least about 6 hours, atleast about 8 hours, at least about 12 hours, at least about 24 hours,at least about 2 days, at least about 3 days, at least about 4 days, atleast about 1 week, at least about 2 weeks, or at least about 3 weeks.

In one aspect, the invention features a pharmaceutically acceptablecomposition comprising the preparation described herein.

In one aspect, the invention features a pharmaceutically acceptablecomposition comprising a liposome described herein.

In one aspect, the invention features a method of treating a mammalcomprising administering to said mammal a therapeutic amount of apharmaceutically acceptable composition, for example, an associationcomplex such as a liposome described herein.

DEFINITIONS

The term “halo” or “halogen” refers to any radical of fluorine,chlorine, bromine or iodine.

The term “alkyl” refers to a hydrocarbon chain that may be a straightchain or branched chain, containing the indicated number of carbonatoms. For example, C₁-C₃₆ alkyl indicates that the group may have from1 to 136 (inclusive) carbon atoms in it. The term “haloalkyl” refers toan alkyl in which one or more hydrogen atoms are replaced by halo, andincludes alkyl moieties in which all hydrogens have been replaced byhalo (e.g., perfluoroalkyl). The terms “arylalkyl” or “aralkyl” refer toan alkyl moiety in which an alkyl hydrogen atom is replaced by an arylgroup. Aralkyl includes groups in which more than one hydrogen atom hasbeen replaced by an aryl group. Examples of “arylalkyl” or “aralkyl”include benzyl, 2-phenylethyl, 3-phenylpropyl, 9-fluorenyl, benzhydryl,and trityl groups.

The term “alkylene” refers to a divalent alkyl, e.g., —CH₂—, —CH₂CH₂—,—CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂, —CH₂CH₂CH₂CH₂CH₂—, and CH₂CH₂CH₂CH₂CH₂CH₂—.

The term “alkenyl” refers to a straight or branched hydrocarbon chaincontaining 2-36 carbon atoms and having one or more double bonds.Examples of alkenyl groups include, but are not limited to, allyl,propenyl, 2-butenyl, 3-hexenyl and 3-octenyl groups. One of the doublebond carbons may optionally be the point of attachment of the alkenylsubstituent. The term “alkynyl” refers to a straight or branchedhydrocarbon chain containing 2-36 carbon atoms and characterized inhaving one or more triple bonds. Examples of alkynyl groups include, butare not limited to, ethynyl, propargyl, and 3-hexynyl. One of the triplebond carbons may optionally be the point of attachment of the alkynylsubstituent.

The term “substituents” refers to a group “substituted” on an alkyl,cycloalkyl, alkenyl, alkynyl, heterocyclyl, heterocycloalkenyl,cycloalkenyl, aryl, or heteroaryl group at any atom of that group. Anyatom can be substituted. Suitable substituents include, withoutlimitation, alkyl (e.g., C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11,C12 straight or branched chain alkyl), cycloalkyl, haloalkyl (e.g.,perfluoroalkyl such as CF₃), aryl, heteroaryl, aralkyl, heteroaralkyl,heterocyclyl, alkenyl, alkynyl, cycloalkenyl, heterocycloalkenyl,alkoxy, haloalkoxy (e.g., perfluoroalkoxy such as OCF₃), halo, hydroxy,carboxy, carboxylate, cyano, nitro, amino, alkyl amino, SO₃H, sulfate,phosphate, methylenedioxy (—O—CH₂—O— wherein oxygens are attached tosame carbon (geminal substitution) atoms), ethylenedioxy, oxo, thioxo(e.g., C═S), imino (alkyl, aryl, aralkyl), S(O)_(n)alkyl (where n is0-2), S(O)_(n) aryl (where n is 0-2), S(O)_(n) heteroaryl (where n is0-2), S(O)_(n) heterocyclyl (where n is 0-2), amine (mono-, di-, alkyl,cycloalkyl, aralkyl, heteroaralkyl, aryl, heteroaryl, and combinationsthereof), ester (alkyl, aralkyl, heteroaralkyl, aryl, heteroaryl), amide(mono-, di-, alkyl, aralkyl, heteroaralkyl, aryl, heteroaryl, andcombinations thereof), sulfonamide (mono-, di-, alkyl, aralkyl,heteroaralkyl, and combinations thereof). In one aspect, thesubstituents on a group are independently any one single, or any subsetof the aforementioned substituents. In another aspect, a substituent mayitself be substituted with any one of the above substituents.

The term “structural isomer” as used herein refers to any of two or morechemical compounds, such as propyl alcohol and isopropyl alcohol, havingthe same molecular formula but different structural formulas.

The term “geometric isomer” or “stereoisomer” as used herein refers totwo or more compounds which contain the same number and types of atoms,and bonds (i.e., the connectivity between atoms is the same), but whichhave different spatial arrangements of the atoms, for example cis andtrans isomers of a double bond, enantiomers, and diasteriomers.

For convenience, the meaning of certain terms and phrases used in thespecification, examples, and appended claims, are provided below. Ifthere is an apparent discrepancy between the usage of a term in otherparts of this specification and its definition provided in this section,the definition in this section shall prevail.

“G,” “C,” “A” and “U” each generally stand for a nucleotide thatcontains guanine, cytosine, adenine, and uracil as a base, respectively.However, it will be understood that the term “ribonucleotide” or“nucleotide” can also refer to a modified nucleotide, as furtherdetailed below, or a surrogate replacement moiety. The skilled person iswell aware that guanine, cytosine, adenine, and uracil may be replacedby other moieties without substantially altering the base pairingproperties of an oligonucleotide comprising a nucleotide bearing suchreplacement moiety. For example, without limitation, a nucleotidecomprising inosine as its base may base pair with nucleotides containingadenine, cytosine, or uracil. Hence, nucleotides containing uracil,guanine, or adenine may be replaced in the nucleotide sequences of theinvention by a nucleotide containing, for example, inosine. Sequencescomprising such replacement moieties are embodiments of the invention.

As used herein, “target sequence” refers to a contiguous portion of thenucleotide sequence of an mRNA molecule formed during the transcriptionof the corresponding gene, including mRNA that is a product of RNAprocessing of a primary transcription product. A target region is asegment in a target gene that is complementary to a portion of the RNAiagent.

As used herein, the term “strand comprising a sequence” refers to anoligonucleotide comprising a chain of nucleotides that is described bythe sequence referred to using the standard nucleotide nomenclature.

As used herein, and unless otherwise indicated, the term“complementary,” when used to describe a first nucleotide sequence inrelation to a second nucleotide sequence, refers to the ability of anoligonucleotide or polynucleotide comprising the first nucleotidesequence to hybridize and form a duplex structure under certainconditions with an oligonucleotide or polynucleotide comprising thesecond nucleotide sequence, as will be understood by the skilled person.Such conditions can, for example, be stringent conditions, wherestringent conditions may include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mMEDTA, 50° C. or 70° C. for 12-16 hours followed by washing. Otherconditions, such as physiologically relevant conditions as may beencountered inside an organism, can apply. The skilled person will beable to determine the set of conditions most appropriate for a test ofcomplementarity of two sequences in accordance with the ultimateapplication of the hybridized nucleotides.

This includes base-pairing of the oligonucleotide or polynucleotidecomprising the first nucleotide sequence to the oligonucleotide orpolynucleotide comprising the second nucleotide sequence over the entirelength of the first and second nucleotide sequence. Such sequences canbe referred to as “fully complementary” with respect to each otherherein. However, where a first sequence is referred to as “substantiallycomplementary” with respect to a second sequence herein, the twosequences can be fully complementary, or they may form one or more, butgenerally not more than 4, 3 or 2 mismatched base pairs uponhybridization, while retaining the ability to hybridize under theconditions most relevant to their ultimate application. However, wheretwo oligonucleotides are designed to form, upon hybridization, one ormore single stranded overhangs, such overhangs shall not be regarded asmismatches with regard to the determination of complementarity. Forexample, an oligonucleotide agent comprising one oligonucleotide 21nucleotides in length and another oligonucleotide 23 nucleotides inlength, wherein the longer oligonucleotide comprises a sequence of 21nucleotides that is fully complementary to the shorter oligonucleotide,may yet be referred to as “fully complementary” for the purposes of theinvention.

“Complementary” sequences, as used herein, may also include, or beformed entirely from, non-Watson-Crick base pairs and/or base pairsformed from non-natural and modified nucleotides, in as far as the aboverequirements with respect to their ability to hybridize are fulfilled.

The terms “complementary”, “fully complementary” and “substantiallycomplementary” herein may be used with respect to the base matchingbetween the sense strand and the antisense strand of an oligonucleotideagent, or between the antisense strand of an oligonucleotide agent and atarget sequence, as will be understood from the context of their use.

As used herein, a polynucleotide which is “substantially complementaryto at least part of” a messenger RNA (mRNA) refers to a polynucleotidewhich is substantially complementary to a contiguous portion of the mRNAof interest. For example, a polynucleotide is complementary to at leasta part of an ApoB mRNA if the sequence is substantially complementary toa non-interrupted portion of a mRNA encoding ApoB.

As used herein, an “oligonucleotide agent” refers to a single strandedoligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid(DNA) or both or modifications thereof, which is antisense with respectto its target. This term includes oligonucleotides composed ofnaturally-occurring nucleobases, sugars and covalent internucleoside(backbone) linkages as well as oligonucleotides havingnon-naturally-occurring portions which function similarly. Such modifiedor substituted oligonucleotides are often preferred over native formsbecause of desirable properties such as, for example, enhanced cellularuptake, enhanced affinity for nucleic acid target and increasedstability in the presence of nucleases.

Oligonucleotide agents include both nucleic acid targeting (NAT)oligonucleotide agents and protein-targeting (PT) oligonucleotideagents. NAT and PT oligonucleotide agents refer to single strandedoligomers or polymers of ribonucleic acid (RNA) or deoxyribonucleic acid(DNA) or both or modifications thereof. This term includesoligonucleotides composed of naturally occurring nucleobases, sugars,and covalent internucleoside (backbone) linkages as well asoligonucleotides having non-naturally-occurring portions that functionsimilarly. Such modified or substituted oligonucleotides are oftenpreferred over native forms because of desirable properties such as, forexample, enhanced cellular uptake, enhanced affinity for nucleic acidtarget, and/or increased stability in the presence of nucleases. NATsdesigned to bind to specific RNA or DNA targets have substantialcomplementarity, e.g., at least 70, 80, 90, or 100% complementary, withat least 10, 20, or 30 or more bases of a target nucleic acid, andinclude antisense RNAs, microRNAs, antagomirs and other non-duplexstructures which can modulate expression. Other NAT oligonucleotideagents include external guide sequence (EGS) oligonucleotides(oligozymes), DNAzymes, and ribozymes. The NAT oligonucleotide agentscan target any nucleic acid, e.g., a miRNA, a pre-miRNA, a pre-mRNA, anmRNA, or a DNA. These NAT oligonucleotide agents may or may not bind viaWatson-Crick complementarity to their targets. PT oligonucleotide agentsbind to protein targets, preferably by virtue of three-dimensionalinteractions, and modulate protein activity. They include decoy RNAs,aptamers, and the like.

While not wishing to be bound by theory, an oligonucleotide agent mayact by one or more of a number of mechanisms, including acleavage-dependent or cleavage-independent mechanism. A cleavage-basedmechanism can be RNAse H dependent and/or can include RISC complexfunction. Cleavage-independent mechanisms include occupancy-basedtranslational arrest, such as can be mediated by miRNAs, or binding ofthe oligonucleotide agent to a protein, as do aptamers. Oligonucleotideagents may also be used to alter the expression of genes by changing thechoice of splice site in a pre-mRNA. Inhibition of splicing can alsoresult in degradation of the improperly processed message, thusdown-regulating gene expression.

The term “double-stranded RNA” or “dsRNA”, as used herein, refers to acomplex of ribonucleic acid molecules, having a duplex structurecomprising two anti-parallel and substantially complementary, as definedabove, nucleic acid strands. The two strands forming the duplexstructure may be different portions of one larger RNA molecule, or theymay be separate RNA molecules. Where separate RNA molecules, such dsRNAare often referred to in the literature as siRNA (“short interferingRNA”). Where the two strands are part of one larger molecule, andtherefore are connected by an uninterrupted chain of nucleotides betweenthe 3′-end of one strand and the 5′ end of the respective other strandforming the duplex structure, the connecting RNA chain is referred to asa “hairpin loop”, “short hairpin RNA” or “shRNA”. Where the two strandsare connected covalently by means other than an uninterrupted chain ofnucleotides between the 3′-end of one strand and the 5′ end of therespective other strand forming the duplex structure, the connectingstructure is referred to as a “linker”. The RNA strands may have thesame or a different number of nucleotides. The maximum number of basepairs is the number of nucleotides in the shortest strand of the dsRNAminus any overhangs that are present in the duplex. In addition to theduplex structure, a dsRNA may comprise one or more nucleotide overhangs.In addition, as used in this specification, “dsRNA” may include chemicalmodifications to ribonucleotides, including substantial modifications atmultiple nucleotides and including all types of modifications disclosedherein or known in the art. Any such modifications, as used in an siRNAtype molecule, are encompassed by “dsRNA” for the purposes of thisspecification and claims.

As used herein, a “nucleotide overhang” refers to the unpairednucleotide or nucleotides that protrude from the duplex structure of adsRNA when a 3′-end of one strand of the dsRNA extends beyond the 5′-endof the other strand, or vice versa. “Blunt” or “blunt end” means thatthere are no unpaired nucleotides at that end of the dsRNA, i.e., nonucleotide overhang. A “blunt ended” dsRNA is a dsRNA that isdouble-stranded over its entire length, i.e., no nucleotide overhang ateither end of the molecule. For clarity, chemical caps or non-nucleotidechemical moieties conjugated to the 3′ end or 5′ end of an siRNA are notconsidered in determining whether an siRNA has an overhang or is bluntended.

The term “antisense strand” refers to the strand of a dsRNA whichincludes a region that is substantially complementary to a targetsequence. As used herein, the term “region of complementarity” refers tothe region on the antisense strand that is substantially complementaryto a sequence, for example a target sequence, as defined herein. Wherethe region of complementarity is not fully complementary to the targetsequence, the mismatches are most tolerated in the terminal regions and,if present, are generally in a terminal region or regions, e.g., within6, 5, 4, 3, or 2 nucleotides of the 5′ and/or 3′ terminus

The term “sense strand,” as used herein, refers to the strand of a dsRNAthat includes a region that is substantially complementary to a regionof the antisense strand.

The terms “silence” and “inhibit the expression of”, in as far as theyrefer to a target gene, herein refer to the at least partial suppressionof the expression of the gene, as manifested by a reduction of theamount of mRNA transcribed from the gene which may be isolated from afirst cell or group of cells in which the gene is transcribed and whichhas or have been treated such that the expression of the gene isinhibited, as compared to a second cell or group of cells substantiallyidentical to the first cell or group of cells but which has or have notbeen so treated (control cells). The degree of inhibition is usuallyexpressed in terms of

${\frac{\left( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{control}\mspace{14mu}{cells}} \right) - \left( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{treated}\mspace{14mu}{cells}} \right)}{\left( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{control}\mspace{14mu}{cells}} \right)} \cdot 100}\%$

Alternatively, the degree of inhibition may be given in terms of areduction of a parameter that is functionally linked to genetranscription, e.g. the amount of protein encoded by the gene which issecreted by a cell, or the number of cells displaying a certainphenotype, e.g apoptosis. In principle, gene silencing may be determinedin any cell expressing the target, either constitutively or by genomicengineering, and by any appropriate assay. However, when a reference isneeded in order to determine whether a given dsRNA inhibits theexpression of the gene by a certain degree and therefore is encompassedby the instant invention, the assay provided in the Examples below shallserve as such reference.

For example, in certain instances, expression of the gene is suppressedby at least about 20%, 25%, 35%, or 50% by administration of thedouble-stranded oligonucleotide of the invention. In some embodiment,the gene is suppressed by at least about 60%, 70%, or 80% byadministration of the double-stranded oligonucleotide of the invention.In some embodiments, the gene is suppressed by at least about 85%, 90%,or 95% by administration of the double-stranded oligonucleotide of theinvention.

As used herein, the terms “treat”, “treatment”, and the like, refer torelief from or alleviation of pathological processes which can bemediated by down regulating a particular gene. In the context of thepresent invention insofar as it relates to any of the other conditionsrecited herein below (other than pathological processes which can bemediated by down regulating the gene), the terms “treat”, “treatment”,and the like mean to relieve or alleviate at least one symptomassociated with such condition, or to slow or reverse the progression ofsuch condition.

As used herein, the phrases “therapeutically effective amount” and“prophylactically effective amount” refer to an amount that provides atherapeutic benefit in the treatment, prevention, or management ofpathological processes which can be mediated by down regulating the geneon or an overt symptom of pathological processes which can be mediatedby down regulating the gene. The specific amount that is therapeuticallyeffective can be readily determined by ordinary medical practitioner,and may vary depending on factors known in the art, such as, e.g. thetype of pathological processes which can be mediated by down regulatingthe gene, the patient's history and age, the stage of pathologicalprocesses which can be mediated by down regulating gene expression, andthe administration of other anti-pathological processes which can bemediated by down regulating gene expression. An effective amount, in thecontext of treating a subject, is sufficient to produce a therapeuticbenefit. The term “therapeutic benefit” as used herein refers toanything that promotes or enhances the well-being of the subject withrespect to the medical treatment of the subject's cell proliferativedisease. A list of nonexhaustive examples of this includes extension ofthe patients life by any period of time; decrease or delay in theneoplastic development of the disease; decrease in hyperproliferation;reduction in tumor growth; delay of metastases; reduction in theproliferation rate of a cancer cell, tumor cell, or any otherhyperproliferative cell; induction of apoptosis in any treated cell orin any cell affected by a treated cell; and/or a decrease in pain to thesubject that can be attributed to the patient's condition.

As used herein, a “pharmaceutical composition” comprises apharmacologically effective amount of an oligonucleotide agent and apharmaceutically acceptable carrier. As used herein, “pharmacologicallyeffective amount,” “therapeutically effective amount” or simply“effective amount” refers to that amount of an RNA effective to producethe intended pharmacological, therapeutic or preventive result. Forexample, if a given clinical treatment is considered effective whenthere is at least a 25% reduction in a measurable parameter associatedwith a disease or disorder, a therapeutically effective amount of a drugfor the treatment of that disease or disorder is the amount necessary toeffect at least a 25% reduction in that parameter.

The term “pharmaceutically acceptable carrier” refers to a carrier foradministration of a therapeutic agent. Such carriers include, but arenot limited to, saline, buffered saline, dextrose, water, glycerol,ethanol, and combinations thereof and are described in more detailbelow. The term specifically excludes cell culture medium.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 depicts a bar graph comparing the efficacy of various ND98compositions.

FIG. 2 depicts a bar graph comparing the efficacy of various ND98compositions.

FIG. 3 depicts a bar graph demonsrating the efficacy of a 6-tailedisomer of ND98.

FIG. 4 depicts a bar graph comparing the efficacy of associationcomplexes prepared using two different procedures.

FIG. 5 depicts various PEG lipid moieties, including those havingvarious chain lengths.

FIG. 6 depicts a bar graph comparing the efficacy of associationcomplexes.

FIG. 7 depicts a bar graph comparing the tolerability of variouscomplexes as the ratio of lipid to siRNA is reduced.

FIG. 8 is a flow chart of a process for making an association complexloaded with nucleic acid.

FIG. 9 are bar graphs depicting the efficacy of siRNAs with two targets,FVII and ApoB.

FIG. 10 is a flow chart of a process for making an association complexloaded with nucleic acid.

FIG. 11 is a bar graph depicting the effect of particle size ofassociation complexes on the efficacy of a nucleic acid in a silencingassay.

FIGS. 12 a and 12 b are bar graphs comparing the serum half life ofnucleic acid therapeutics in unformulated and formulated forms.

FIG. 13 is a bar graph comparing the efficacy of association complexeshaving PEG lipids with varied chain lengths.

DETAILED DESCRIPTION

Lipid preparations and delivery systems useful to administer nucleicacid based therapies such as siRNA are described herein.

Cationic Lipid Compounds and Lipid Preparations

Polyamine Lipid Preparations

Applicants have discovered that certain polyamine lipid moieties providedesirable properties for administration of nucleic acids, such as siRNA.For example, in some embodiments, a lipid moiety is complexed with aFactor VII-targeting siRNA and administered to an animal such as amouse. The level of secreted serum Factor VII is then quantified (24 hpost administration), where the degree of Factor VII silencing indicatesthe degree of in vivo siRNA delivery. Accordingly, lipids providingenhanced in vivo delivery of a nucleic acid such as siRNA are preferred.In particular, Applicants have discovered polyamines havingsubstitutions described herein can have desirable properties fordelivering siRNA, such as bioavailability, biodegradability, andtolerability.

In one embodiment, a lipid preparation includes a polyamine moietyhaving a plurality of substituents, such as acrylamide or acrylatesubstituents attached thereto. For example, a lipid moiety can include apolyamine moiety as provided below,

where one or more of the hydrogen atoms are substituted, for examplewith a substituent including a long chain alkyl, alkenyl, or alkynylmoiety, which in some embodiments is further substituted. X^(a) andX^(b) are alkylene moieties. In some embodiments, X^(a) and X^(b) havethe same chain length, for example X^(a) and X^(b) are both ethylenemoieties. In other embodiments X^(a) and X^(b) are of differing chainlengths. In some embodiments, where the polyamine includes a pluralityof X′ moieties, X′ can vary with one or more occurrences. For example,where the polyamine is spermine, X′ in one occurrence is propylene,X^(a) in another occurrence is butylenes, and X^(b) is propylene.

Applicants have discovered that in some instances it is desirable tohave a relatively high degree of substitution on the polyamine Forexample, in some embodiments, Applicants have discovered that polyaminepreparations where at least 80% (e.g., at least about 85%, at leastabout 90%, at least about 95%, at least about 97%, at least about 98%,at least about 99%, or substantially all) of the polyamines in thepreparation have at least n+2 of the hydrogens substituted with asubstituent provide desirable properties, for example for use inadministering a nucleic acid such as siRNA.

In some instances it is desirable (preferably) to have one or more ofhetero atoms present on the substituent on the nitrogen of polyamine

In some embodiments, a preparation comprises a compound of formula (I)or a pharmaceutically acceptable salt thereof,

each X^(a) and X^(b), for each occurrence, is independently C₁₋₆alkylene; n is 0, 1, 2, 3, 4, or 5; each R is independently H,

wherein at least n+2 of the R moieties in at least about 80% of themolecules of the compound of formula (I) in the preparation are not H; mis 1, 2, 3 or 4; Y is O, NR², or S; R¹ is alkyl alkenyl or alkynyl; eachof which is optionally substituted; and R² is H, alkyl alkenyl oralkynyl; each of which is optionally substituted; provided that, if n=0,than at least n+3 of the R moieties are not H.

As noted above, the preparation includes molecules containingsymmetrical as well as asymmetrical polyamine derivatives. Accordingly,X^(a) is independent for each occurrence and X^(b) is independent ofX^(a). For example, where n is 2, X^(a) can either be the same for eachoccurrence or can be different for each occurrence or can be the samefor some occurrences and different for one or more other occurrences.X^(b) is independent of X^(a) regardless of the number of occurrences ofX′ in each polyamine derivative. X′, for each occurrence and independentof X^(b), can be methylene, ethylene, propylene, butylene, pentylene, orhexylene. Exemplary polyamine derivatives include those polyaminesderived from N¹,N^(1′)-(ethane-1,2-diyl)diethane-1,2-diamine,ethane-1,2-diamine, propane-1,3-diamine, spermine, spermidine,putrecine, and N¹-(2-Aminoethyl)-propane-1,3-diamine. Preferredpolyamine derivatives include propane-1,3-diamine andN¹,N^(1′)-(ethane-1,2-diyl)diethane-1,2-diamine.

The polyamine of formula (I) is substituted with at least n+2 R moietiesthat are not H. In general, each non-hydrogen R moiety includes analkyl, alkenyl, or alkynyl moiety, which is optionally substituted withone or more substituents, attached to a nitrogen of the polyaminederivative via a linker. Suitable linkers include amides, esters,thioesters, sulfones, sulfoxides, ethers, amines, and thioethers. Inmany instances, the linker moiety is bound to the nitrogen of thepolyamine via an alkylene moiety (e.g., methylene, ethylene, propylene,or butylene). For example, an amide or ester linker is attached to thenitrogen of the polyamine through a methylene or ethylene moiety.

Examples of preferred amine substituents are provided below:

In instances where the amine is bound to the linker-R¹ portion via anethylene group, a 1,4 conjugated precursor acrylate or acrylamide can bereacted with the polyamine to provide the substituted polyamine. Ininstances where the amine is bound to the linker-R¹ portion via amethylene group, an amide or ester including an alpha-halo substituent,such as an alpha-chloro moiety, can be reacted with the polyamine toprovide the substituted polyamine. In preferred embodiments, R² is H.

R moieties that are not H, all require an R¹ moiety as provided above.In general, the R¹ moiety is a long chain moiety, such as C₆-C₃₂ alkyl,C₆-C₃₂ alkenyl, or C₆-C₃₂ alkynyl.

In some preferred embodiments, R¹ is an alkyl moiety. For example R¹ isC₁₀-C₁₈ alkyl, such as C₁₂ alkyl. Examples of especially preferred Rmoieties are provided below.

The preparations including a compound of formula (I) can be mixtures ofa plurality of compounds of formula (I). For example, the preparationcan include a mixture of compounds of formula (I) having varying degreesof substitution on the polyamine moiety. However, the preparationsdescribed herein are selected such that at least n+2 of the R moietiesin at least about 80% (e.g., at least about 85%, at least about 90%, atleast about 95%, at least about 97%, at least about 98%, at least about99%, or substantially all) of the molecules of the compound of formula(I) in the preparation are not H.

In some embodiments, a preparation includes a polyamine moiety havingtwo amino groups wherein in at least 80% (e.g., at least about 85%, atleast about 90%, at least about 95%, at least about 97%, at least about98%, at least about 99%, or substantially all) of the molecules offormula (I) in the mixture are substituted with three R moieties thatare not H. Exemplary compounds of formula (I) are provided below.

In some preferred embodiments R is

In some preferred embodiments, R¹ is C₁₀-C₁₈ alkyl, or C₁₀-C₃₀ alkenyl.

In some embodiments, a preparation includes a polyamine moiety havingthree or four (e.g., four) amino groups wherein at least n+2 of the Rmoieties in at least about 80% (e.g., at least about 85%, at least about90%, at least about 95%, at least about 97%, at least about 98%, atleast about 99%, or substantially all) of the molecules of formula (I)are not H. Exemplary compounds of formula (I) having 4 amino moietiesare provided below.

Examples of polyamine moiety where all (i.e., n+4) R moieties are not Hare below:

In some preferred embodiments R is

In some preferred embodiments, R¹ is C₁₀-C₁₈ alkyl (e.g., C₁₂ alkyl), orC₁₀-C₃₀ alkenyl.

Examples of polyamine moieties where five (i.e., n+3) R moieties are notH are provided below:

In some preferred embodiments R is

In some preferred embodiments, R¹ is C₁₀-C₁₈ alkyl (e.g., C₁₂ alkyl), orC₁₀-C₃₀ alkenyl.

Examples of polyamine moieties where four (i.e, n+2) R moieties are notH are provided below:

In some preferred embodiments R is

In some preferred embodiments, R¹ is C₁₀-C₁₈ alkyl (e.g., C₁₂ alkyl), orC₁₀-C₃₀ alkenyl.

In some preferred embodiments, the polyamine is a compound of isomer (1)or (2) below, preferably a compound of isomer (1)

In some embodiments, the preparation including a compound of formula (I)includes a mixture of molecules having formula (I). For example, themixture can include molecules having the same polyamine core butdiffering R substituents, such as differing degrees of R substituentsthat are not H.

In some embodiments, a preparation described herein includes a compoundof formula (I) having a single polyamine core wherein each R of thepolyamine core is either R or a single moiety such as

The preparation, therefore includes a mixture of molecules havingformula (I), wherein the mixture is comprised of either polyaminecompounds of formula (I) having a varied number of R moieties that are Hand/or a polyamine compounds of formula (I) having a single determinednumber of R moieties that are not H where the compounds of formula (I)are structural isomers of the polyamine, such as the structural isomersprovided above.

In some preferred embodiments the preparation includes molecules offormula (I) such that at least 80% (e.g., at least about 85%, at leastabout 90%, at least about 95%, at least about 97%, at least about 98%,at least about 99%, or substantially all) of the molecules are a singlestructural isomer.

In some embodiments, the preparation includes a mixture of two or morecompounds of formula (I). In some embodiments, the preparation is amixture of structural isomers of the same chemical formula. In someembodiments, the preparation is a mixture of compounds of formula (I)where the compounds vary in the chemical nature of the R substituents.For example, the preparation can include a mixture of the followingcompounds:

wherein n is 0 and each R is independently H or

wherein n is 2 and each R is independently H or

In some embodiments, the compound of formula (I) is in the form of asalt, such as a pharmaceutically acceptable salt. A salt, for example,can be formed between an anion and a positively charged substituent(e.g., amino) on a compound described herein. Suitable anions includefluoride, chloride, bromide, iodide, sulfate, bisulfate, nitrate,phosphate, citrate, methanesulfonate, trifluoroacetate, acetate,fumarate, oleate, valerate, maleate, oxalate, isonicotinate, lactate,salicylate, tartrate, tannate, pantothenate, bitartrate, ascorbate,succinate, gentisinate, gluconate, glucaronate, saccharate, formate,benzoate, glutamate, ethanesulfonate, benzenesulfonate, p-toluensulfonate, and pamoate. In some preferred embodiments, thecompound of formula (I) is a hydrohalide salt, such as a hydrochloridesalt.

Compounds of formula (I) can also be present in the form of hydrates(e.g., (H₂O)_(n)) and solvates, which are included herewith in thedisclosure.

Biocleavable Cationic Lipids

Applicants have discovered that certain cationic lipids that include oneor more biocleavable moieties can be used as a component in anassociation complex, such as a liposome, for the delivery of nucleicacid therapies (e.g., dsRNA). For example, disclosed herein are cationiclipids that are subject to cleavage in vivo, for example, via an enzymesuch as an esterase, an amidase, or a disulfide cleaving enzyme. In someinstances, the lipid is cleaved chemically, for example by hydrolysis ofan acid labile moiety such as an acetal or ketal. In some embodiments,the lipid includes a moiety that is hydrolyzed in vitro and then subjectto enzymatic cleavage by one or more of an esterase, amidase, or adisulfide cleaving enzyme. This can happen in vesicular compartments ofthe cell such as endosomes. Another acid sensitive cleavable linkage isβ-thiopropionate linkage which is cleaved in the acidic environment ofendosomes (Jeong et al. Bioconjugate chem. 2003, 4, 1426).

In some embodiments, the invention features a compound of formula (X) ora pharmaceutically acceptable salt thereof, wherein

wherein

R¹ and R² are each independently H, C₁-C₆ alkyl, optionally substitutedwith 1-4 R⁵, C₂-C₆ alkenyl, optionally substituted with 1-4 R⁵, orC(NR⁶)(NR⁶)₂;

R³ and R⁴ are each independently alkyl, alkenyl, alkynly, each of whichis optionally substituted with fluoro, chloro, bromo, or iodo;

L¹ and L² are each independently —NR⁶C(O)—, —C(O)NR⁶—, —OC(O)—, —C(O)O—,—S—S—, —N(R⁶)C(O)N(R⁶)—, —OC(O)N(R⁶)—, —N(R⁶)C(O)O—, —O—N═O—,OR—OC(O)NH; or

L¹-R³ and L²-R⁴ can be taken together to form an acetal or a ketal;

R⁵ is fluoro, chloro, bromo, iodo, —OR⁷, —N(R⁸)(R⁹), —CN, SR¹⁰, S(O)R¹⁰,S(O)₂R¹⁰

R⁶ is H, C₁-C₆ alkyl,

R⁷ is H or C₁-C₆ alkyl;

each R⁸ and R⁹ are independently H or C₁-C₆ alkyl;

R¹⁰ is H or C₁-C₆ alkyl;

m is 1, 2, 3, 4, 5, or 6;

n is 0, 1, 2, 3, 4, 5, or 6;

and pharmaceutically acceptable salts thereof.

In some embodiments, R¹ is H, a lower alkyl, such as methyl, ethyl,propyl, or isopropyl, or a substituted alkyl, such as 2-hydroxyethyl.

In some embodiments, R² is H or a lower alkyl, such as methyl, ethyl,propyl, or isopropyl.

In some embodiments, R¹ or R² form a quanadine moiety with the nitrogenof formula (X).

L¹-R³ and L²-R⁴ or the combination thereof provide at least one moietythat is cleaved in vivo. In some embodiments, both L¹-R³ and L²-R⁴ arebiocleavable. For example, both L¹-R³ and L²-R⁴ are independentlysubject to enzymatic cleavage (e.g., by an esterase, amidase, or adisulfide cleaving enzyme). In some embodiments, both L¹ and L² are thesame chemical moiety such as an ester, amide or disulfide. In otherinstances, L¹ and L² are different, for example, one of L¹ or L² is anester an the other of L¹ or L² is a disulfide.

In some embodiments, L¹-R³ and L²-R⁴ together form an acetal or ketalmoiety, which is hydrolyzed in vivo.

In some embodiments, one of L¹-R³ or L²-R⁴ is subject to enzymaticcleavage. For example, one of L¹-R³ or L²-R⁴ is cleaved in vivo,providing a free hydroxyl moiety or free amine on the lipid, whichbecomes available to chemically react with the remaining L¹-R³ or L²-R⁴moiety. Exemplary embodiments are provided below:

In some preferred embodiments, a carbamate or urea moiety is included incombination with an amide, ester or disulfide moiety. For example, thelipid includes an ester moiety, which upon cleavage (e.g., enzymaticcleavage) becomes available to chemically react with the carbamate orurea moiety. Some preferred combinations of L¹ and L² include twoamides, two esters, an amide and an ester, two disulfides, an amide anda disulfide, an ester and a disulfide, a carbamate and a disulfide, anda urea and a disulfide. Exemplary compounds are provided below:Amide and Ester Linkages with Z Configuration (Two Double Bonds)

-   R′=H, Me, Et, propyl, isopropyl or 2-hydroxyethyl and R″=H; l=1 to    6, m=1-8, n=1-10-   R′=H, Me, Et, propyl, isopropyl or 2-hydroxyethyl and R″=Me; l=1 to    6, m=1-8, n=1-10-   R′=H, Me, Et, propyl, isopropyl or 2-hydroxyethyl and R″=Et; l=1 to    6, m=1-8, n=1-10-   R′=H, Me, Et, propyl, isopropyl or 2-hydroxyethyl and R″=propyl; l=1    to 6, m=1-8, n=1-10-   R′=H, Me, Et, propyl, isopropyl or 2-hydroxyethyl and R″=isopropyl;    l=1 to 6, m=1-8, n=1-10    Amide Ester Linkage with Z Configuration (Three Double Bonds)

-   R′=H, Me, Et, propyl, isopropyl or 2-hydroxyethyl and R″=H; l=1 to    6, m=1-8, n=1-10-   R′=H, Me, Et, propyl, isopropyl or 2-hydroxyethyl and R″=Me; l=1 to    6, m=1-8, n=1-10-   R′=H, Me, Et, propyl, isopropyl or 2-hydroxyethyl and R″=Et; l=1 to    6, m=1-8, n=1-10-   R′=H, Me, Et, propyl, isopropyl or 2-hydroxyethyl and R″=propyl; l=1    to 6, m=1-8, n=1-10-   R′=H, Me, Et, propyl, isopropyl or 2-hydroxyethyl and R″=isopropyl;    l=1 to 6, m=1-8, n=1-10    Amides and Ester Linkages with E Configuration (Two Double Bonds)

-   R′=H, Me, Et, propyl, isopropyl or 2-hydroxyethyl and R″=H; l=1 to    6, m=1-8, n=1-10-   R′=H, Me, Et, propyl, isopropyl or 2-hydroxyethyl and R″=Me; l=1 to    6, m=1-8, n=1-10-   R′=H, Me, Et, propyl, isopropyl or 2-hydroxyethyl and R″=Et; l=1 to    6, m=1-8, n=1-10-   R′=H, Me, Et, propyl, isopropyl or 2-hydroxyethyl and R″=propyl; l=1    to 6, m=1-8, n=1-10-   R′=H, Me, Et, propyl, isopropyl or 2-hydroxyethyl and R″=isopropyl;    l=1 to 6, m=1-8, n=1-10    Amides and Ester Linkages with E Configuration (Three Double Bonds)

-   R′=H, Me, Et, propyl, isopropyl or 2-hydroxyethyl and R″=H; l=1 to    6, m=1-8, n=1-10-   R′=H, Me, Et, propyl, isopropyl or 2-hydroxyethyl and R″=Me; l=1 to    6, m=1-8, n=1-10-   R′=H, Me, Et, propyl, isopropyl or 2-hydroxyethyl and R″=Et; l=1 to    6, m=1-8, n=1-10-   R′=H, Me, Et, propyl, isopropyl or 2-hydroxyethyl and R″=propyl; l=1    to 6, m=1-8, n=1-10-   R′=H, Me, Et, propyl, isopropyl or 2-hydroxyethyl and R″=isopropyl;    l=1 to 6, m=1-8, n=1-10    Disulfide Linkages

-   R′═H, Me, Et, propyl, isopropyl or 2-hydroxyethyl and R″═H; l=1 to    6, m=6-28-   R′=H, Me, Et, propyl, isopropyl or 2-hydroxyethyl and R″=Me; l=1 to    6, m=6-28-   R′=H, Me, Et, propyl, isopropyl or 2-hydroxyethyl and R″=Et; l=1 to    6, m=6-28-   R′=H, Me, Et, propyl, isopropyl or 2-hydroxyethyl and R″=propyl; l=1    to 6, m=6-28-   R′=H, Me, Et, propyl, isopropyl or 2-hydroxyethyl and R″=isopropyl;    l=1 to 6, m=6-28    Disulfide Linkages with Unsaturated Alkyl Chains, E and Z    Configuration

-   R′=H, Me, Et, propyl, isopropyl or 2-hydroxyethyl and R″=H; l=1 to    6, m=1-8, n=1-10-   R′=H, Me, Et, propyl, isopropyl or 2-hydroxyethyl and R″=Me; l=1 to    6, m=1-8, n=1-10-   R′=H, Me, Et, propyl, isopropyl or 2-hydroxyethyl and R″=Et; l=1 to    6, m=1-8, n=1-10-   R′=H, Me, Et, propyl, isopropyl or 2-hydroxyethyl and R″=propyl; l=1    to 6, m=1-8, n=1-10-   R′=H, Me, Et, propyl, isopropyl or 2-hydroxyethyl and R″=isopropyl;    l=1 to 6, m=1-8, n=1-10    Amide and Disulfide Linkages with Saturated and Unsaturated Alkyl    Chains

-   R′=H, Me, Et, propyl, isopropyl or 2-hydroxyethyl and R″=H; l=1 to    6, m=6-28-   R′=H, Me, Et, propyl, isopropyl or 2-hydroxyethyl and R″=Me; l=1 to    6, m=6-28-   R′=H, Me, Et, propyl, isopropyl or 2-hydroxyethyl and R″=Et; l=1 to    6, m=6-28-   R′=H, Me, Et, propyl, isopropyl or 2-hydroxyethyl and R″=propyl; l=1    to 6, m=6-28-   R′=H, Me, Et, propyl, isopropyl or 2-hydroxyethyl and R″=isopropyl;    l=1 to 6, m=6-28

-   R′=H, Me, Et, propyl, isopropyl or 2-hydroxyethyl and R″=H; l=1 to    6, m=1-8, n=1-10-   R′=H, Me, Et, propyl, isopropyl or 2-hydroxyethyl and R″=Me; l=1 to    6, m=1-8, n=1-10-   R′=H, Me, Et, propyl, isopropyl or 2-hydroxyethyl and R″=Et; l=1 to    6, m=1-8, n=1-10-   R′=H, Me, Et, propyl, isopropyl or 2-hydroxyethyl and R″=propyl; l=1    to 6, m=1-8, n=1-10-   R′=H, Me, Et, propyl, isopropyl or 2-hydroxyethyl and R″=isopropyl;    l=1 to 6, m=1-8, n=1-10    Ester and Disulfide Linkages with Saturated and Unsaturated Alkyl    Chains

-   R′=H, Me, Et, propyl, isopropyl or 2-hydroxyethyl and R″=H; l=1 to    6, m=6-28-   R′=H, Me, Et, propyl, isopropyl or 2-hydroxyethyl and R″=Me; l=1 to    6, m=6-28-   R′=H, Me, Et, propyl, isopropyl or 2-hydroxyethyl and R″=Et; l=1 to    6, m=6-28-   R′=H, Me, Et, propyl, isopropyl or 2-hydroxyethyl and R″=propyl; l=1    to 6, m=6-28-   R′=H, Me, Et, propyl, isopropyl or 2-hydroxyethyl and R″=isopropyl;    l=1 to 6, m=6-28

-   R′=H, Me, Et, propyl, isopropyl or 2-hydroxyethyl and R″=H; l=1 to    6, m=1-8, n=1-10-   R′=H, Me, Et, propyl, isopropyl or 2-hydroxyethyl and R″=Me; l=1 to    6, m=1-8, n=1-10-   R′=H, Me, Et, propyl, isopropyl or 2-hydroxyethyl and R″=Et; l=1 to    6, m=1-8, n=1-10-   R′=H, Me, Et, propyl, isopropyl or 2-hydroxyethyl and R″=propyl; l=1    to 6, m=1-8, n=1-10-   R′=H, Me, Et, propyl, isopropyl or 2-hydroxyethyl and R″=isopropyl;    l=1 to 6, m=1-8, n=1-10    Carbamate or Urea and Disulfide Linkages with Alkyl Chains

-   R′=H, Me, Et, propyl, isopropyl or 2-hydroxyethyl and R″=H; l=1 to    6, m=6-28-   R′=H, Me, Et, propyl, isopropyl or 2-hydroxyethyl and R″=Me; l=1 to    6, m=6-28-   R′=H, Me, Et, propyl, isopropyl or 2-hydroxyethyl and R″=Et; l=1 to    6, m=6-28-   R′=H, Me, Et, propyl, isopropyl or 2-hydroxyethyl and R″=propyl; l=1    to 6, m=6-28-   R′=H, Me, Et, propyl, isopropyl or 2-hydroxyethyl and R″=isopropyl;    l=1 to 6, m=6-28    Carbamate or Urea and Disulfide Linkages with Unsaturated Alkyl    Chains

-   R′=H, Me, Et, propyl, isopropyl or 2-hydroxyethyl and R″=H; l=1 to    6, m=6-28-   R′=H, Me, Et, propyl, isopropyl or 2-hydroxyethyl and R″=Me; l=1 to    6, m=6-28-   R′=H, Me, Et, propyl, isopropyl or 2-hydroxyethyl and R″=Et; l=1 to    6, m=6-28-   R′=H, Me, Et, propyl, isopropyl or 2-hydroxyethyl and R″=propyl; l=1    to 6, m=6-28-   R′=H, Me, Et, propyl, isopropyl or 2-hydroxyethyl and R″=isopropyl;    l=1 to 6, m=6-28    Carbamate or Urea and Disulfide Linkages with Unsaturated Alkyl    Chains

-   R′=H, Me, Et, propyl, isopropyl or 2-hydroxyethyl and R″=H; l=1 to    6, m=6-28-   R′=H, Me, Et, propyl, isopropyl or 2-hydroxyethyl and R″=Me; l=1 to    6, m=6-28-   R′=H, Me, Et, propyl, isopropyl or 2-hydroxyethyl and R″=Et; l=1 to    6, m=6-28-   R′=H, Me, Et, propyl, isopropyl or 2-hydroxyethyl and R″=propyl; l=1    to 6, m=6-28-   R′=H, Me, Et, propyl, isopropyl or 2-hydroxyethyl and R″=isopropyl;    l=1 to 6, m=6-28    Carbamate and Urea Linkages with Unsaturated Alkyl Chains

-   R′=H, Me, Et, propyl, isopropyl or 2-hydroxyethyl and R″=H; l=1 to    6, m=1-10, n=1-10-   R′=H, Me, Et, propyl, isopropyl or 2-hydroxyethyl and R″=Me; l=1 to    6, m=1-10, n=1-10-   R′=H, Me, Et, propyl, isopropyl or 2-hydroxyethyl and R″=Et; l=1 to    6, m=1-10, n=1-10-   R′=H, Me, Et, propyl, isopropyl or 2-hydroxyethyl and R″=propyl; l=1    to 6, m=1-10, n=1-10-   R′=H, Me, Et, propyl, isopropyl or 2-hydroxyethyl and R″=isopropyl;    l=1 to 6, m=1-10, n=1-10

In some embodiments, the lipid includes an oxime or hydrazone, which canundergo acidic cleavage.

R³ and R⁴ are generally long chain hydrophobic moieties, such as alkyl,alkenyl, or alkynyl. In some embodiments, R³ or R⁴ are substituted witha halo moiety, for example, to provide a perfluoroalkyl orperfluoroalkenyl moiety. Each of R³ and R⁴ are independent of eachother. In some embodiments, both of R³ and R⁴ are the same. In someembodiments, R³ and R⁴ are different.

In some embodiments R³ and/or R⁴ are alkyl. For example one or both ofR³ and/or R⁴ are C₆ to C₃₀ alkyl, e.g., C₁₀ to C₂₆ alkyl, C₁₂ to C₂₀alkyl, or C₁₂ alkyl.

In some embodiments, R³ and/or R⁴ are alkenyl. In some preferredembodiments, R³ and/or R⁴ include 2 or 3 double bonds. For example R³and/or R⁴ includes 2 double bonds or R³ and/or R⁴ includes 3 doublebonds. The double bonds can each independently have a Z or Econfiguration. Exemplary alkenyl moieties are provided below:

wherein x is an integer from 1 to 8; and y is an integer from 1-10. Insome preferred embodiments, R³ and/or R⁴ are C₆ to C₃₀ alkenyl, e.g.,C₁₀ to C₂₆ alkenyl, C₁₂ to C₂₀ alkenyl, or C₁₋₇ alkenyl, for examplehaving two double bonds, such as two double bonds with Z configuration.R³ and/or R⁴ can be the same or different. In some preferredembodiments, R³ and R⁴ are the same.

In some embodiments, R³ and/or R⁴ are alkynyl. For example C₆ to C₃₀alkynyl, e.g., C₁₀ to C₂₆ alkynyl, C₁₂ to C₂₀ alkynyl. R³ and/or R⁴ canhave from 1 to 3 triple bonds, for example, one, two, or three triplebonds.

In some embodiments, the compound of formula (X) is in the form of asalt, such as a pharmaceutically acceptable salt. A salt, for example,can be formed between an anion and a positively charged substituent(e.g., amino) on a compound described herein. Suitable anions includefluoride, chloride, bromide, iodide, sulfate, bisulfate, nitrate,phosphate, citrate, methanesulfonate, trifluoroacetate, acetate,fumarate, oleate, valerate, maleate, oxalate, isonicotinate, lactate,salicylate, tartrate, tannate, pantothenate, bitartrate, ascorbate,succinate, gentisinate, gluconate, glucaronate, saccharate, formate,benzoate, glutamate, ethanesulfonate, benzenesulfonate, p-toluensulfonate, and pamoate. In some preferred embodiments, thecompound of formula (X) is a hydrohalide salt, such as a hydrochloridesalt.

Compounds of formula (X) can also be present in the form of hydrates(e.g., (H₂O)_(n)) and solvates, which are included herewith in thedisclosure.

PEG-Lipid Compounds

Applicants have discovered that certain PEG containing lipid moietiesprovide desirable properties for administration of a nucleic acid agentsuch as single stranded or double stranded nucleic acid, for examplesiRNA. For example, when a PEG containing lipid, such as a lipiddescribed herein, is formulated into an association complex with anucleic acid moiety, such as siRNA and administered to a subject, thelipid provides enhanced delivery of the nucleic acid moiety. Thisenhanced delivery can be determined, for example, by evaluation in agene silencing assay such as silencing of FVII. In particular,Applicants have discovered the PEG-lipids of formula (XV) can havedesirable properties for the delivery of siRNA, including improvedbioavailability, diodegradability, and tolerability.

In some embodiment, the PEG is attached via a linker moiety to astructure including two hydrophobic moieties, such as a long chaninalkyl moiety. Exemplary PEG-lipids are provided above, for example,those encompassed by formula (XV), (XV′), and (XVI). In some preferredembodiments, the PEG-lipid has the structure below:

wherein the preferred stereochemistry of the chiral center is ‘R’ andthe repeating PEG moiety has a total average molecular weight of about2000 daltons.

In some embodiments, a PEG lipid described herein is conjugated to atargeting moiety, e.g., a glycosyl moiety such as a

In some embodiments, the targeting moiety is attached to the PEG lipidthrough a linker, for example a linker described herein. Exemplarytargeted PEG lipid compounds are compounds of formula (XXI), (XXI′),(XXII), and (XXII′) described herein. Methods of making such lipids aredescribed, for example, in Examples 42 and 43.Methods of Making Cationic Lipid Compounds and Cationic Lipid ContainingPreparations

The compounds described herein can be obtained from commercial sources(e.g., Asinex, Moscow, Russia; Bionet, Camelford, England; ChemDiv,SanDiego, Calif.; Comgenex, Budapest, Hungary; Enamine, Kiev, Ukraine;IF Lab, Ukraine; Interbioscreen, Moscow, Russia; Maybridge, Tintagel,UK; Specs, The Netherlands; Timtec, Newark, Del.; Vitas-M Lab, Moscow,Russia) or synthesized by conventional methods as shown below usingcommercially available starting materials and reagents.

Methods of Making Polyamine Lipids

In some embodiments, a compound of formula (I) can be made by reacting apolyamine of formula (III) as provided below

wherein X^(a), X^(b), and n are defined as abovewith a 1,4 conjugated system of formula (IV)

wherein Y and R¹ are defined as aboveto provide a compound of formula (I).

In some embodiments, the compounds of formula (III) and (IV) are reactedtogether neat (i.e., free of solvent). For example, the compounds offormula (III) and (IV) are reacted together neat at elevated temperature(e.g., at least about 60° C., at least about 65° C., at least about 70°C., at least about 75° C., at least about 80° C., at least about 85° C.,or at least about 90° C.), preferably at about 90° C.

In some embodiments, the compounds of formula (III) and (IV) are reactedtogether with a solvent (e.g., a polar aprotic solvent such asacetonitrile or DMF). For example, the compounds of formula (III) and(IV) are reacted together in solvent at an elevated temperature fromabout 50° C. to about 120° C.

In some embodiments, the compounds of formula (III) and (IV) are reactedtogether in the presence of a radical quencher or scavenger (e.g.,hydroquinone). The reaction conditions including a radical quencher canbe neat or in a solvent e.g., a polar aprotic solvent such asacetonitrile or DMF. The reaction can be at an elevated temperature(e.g., neat at an elevated temperature such as 90° C. or with solvent atan elevated temperature such as from about 50° C. to about 120° C.). Theterm “radical quencher” or “radical scavenger” as used herein refers toa chemical moiety that can absorb free radicals in a reaction mixture.Examples of radical quenchers/scavengers include hydroquinone, ascorbicacid, cresols, thiamine, 3,5-Di-tert-butyl-4-hydroxytoluene,tert-Butyl-4-hydroxyanisole and thiol containing moieties.

In some embodiments, the compounds of formula (III) and (IV) are reactedtogether in the presence of a reaction promoter (e.g., water or aMichael addition promoter such as acetic acid, boric acid, citric acid,benzoic acid, tosic acid, pentafluorophenol, picric acid aromatic acids,salts such as bicarbonate, bisulphate, mono and di-hydrogen phophates,phenols, perhalophenols, nitrophenols, sulphonic acids, PTTS, etc.),preferably boric acid such as a saturated aqueous boric acid. Thereaction conditions including a reaction promoter can be neat or in asolvent e.g., a polar aprotic solvent such as acetonitrile or DMF. Thereaction can be at an elevated temperature (e.g., neat at an elevatedtemperature such as 90° C. or with solvent at an elevated temperaturesuch as from about 50° C. to about 120° C.). The term “reactionpromoter” as used herein refers to a chemical moiety that, when used ina reaction mixture, accelerates/enhances the rate of reaction.

The ratio of compounds of formula (III) to formula (IV) can be varied,providing variability in the substitution on the polyamine of formula(III). In general, polyamines having at least about 50% of the hydrogenmoieties substituted with a non-hydrogen moiety are preferred.Accordingly, ratios of compounds of formula (III)/formula (IV) areselected to provide for products having a relatively high degree ofsubstitution of the free amine (e.g., at least about 50%, at least about55%, at least about 60%, at least about 65%, at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 97%, at least about 99%, orsubstantially all). In some preferred embodiments n is 0 in thepolyamine of formula (III), and the ratio of compounds of formula (III)to compounds of formula (IV) is from about 1:3 to about 1:5, preferableabout 1:4. In some preferred embodiments, n is 2 in the polyamine offormula (III), and the ratio of compound of formula (III) to compoundsof formula (IV) is from about 1:3 to about 1:6, preferably about 1:5.

In some embodiments, the compounds of formula (III) and formula (IV) arereacted in a two step process. For example, the first step processincludes a reaction mixture having from about 0.8 about 1.2 molarequivalents of a compound of formula (III), with from about 3.8 to about4.2 molar equivalents of a compound of formula (IV) and the second stepprocess includes addition of about 0.8 to 1.2 molar equivalent ofcompound of formula (IV) to the reaction mixture.

Upon completion of the reaction, one or more products having formula (I)can be isolated from the reaction mixture. For example, a compound offormula (I) can be isolated as a single product (e.g., a singlestructural isomer) or as a mixture of product (e.g., a plurality ofstructural isomers and/or a plurality of compounds of formula (I)). Insome embodiments, one or more reaction products can be isolated and/orpurified using chromatography, such as flash chromatography, gravitychromatography (e.g., gravity separation of isomers using silica gel),column chromatography (e.g., normal phase HPLC or RPHPLC), or moving bedchromatography. In some embodiments, a reaction product is purified toprovide a preparation containing at least about 80% of a singlecompound, such as a single structural isomer (e.g., at least about 85%,at least about 90%, at least about 95%, at least about 97%, at leastabout 99%).

In some embodiments, a free amine product is treated with an acid suchas HCl to prove an amine salt of the product (e.g., a hydrochloridesalt). In some embodiments a salt product provides improved properties,e.g., for handling and/or storage, relative to the corresponding freeamine product. In some embodiments, a salt product can prevent or reducethe rate of formation of breakdown product such as N-oxide orN-carbonate formation relative to the corresponding free amine. In someembodiments, a salt product can have improved properties for use in atherapeutic formulation relative to the corresponding free amine.

In some embodiments, the reaction mixture is further treated, forexample, to purify one or more products or to remove impurities such asunreacted starting materials. In some embodiments the reaction mixtureis treated with an immobilized (e.g., polymer bound) thiol moiety, whichcan trap unreacted acrylamide. In some embodiments, an isolated productcan be treated to further remove impurities, e.g., an isolated productcan be treated with an immobilized thiol moiety, trapping unreactedacrylamide compounds.

In some embodiments a reaction product can be treated with animmobilized (e.g., polymer bound) isothiocyanate. For example, areaction product including tertiary amines can be treated with animmobilized isothiocyanate to remove primary and/or secondary aminesfrom the product.

In some embodiments, a compound of formula (I) can be made by reacting apolyamine of formula (III) as provided below

wherein X^(a), X^(b), and n are defined as abovewith a compound of formula (VI)),

wherein Q is Cl, Br, or I, and Y and R¹ are as defined above.

In some embodiments, the compound of formula (III) and formula (VI) arereacted together neat. In some embodiments, the compound of formula(III) and formula (VI) are reacted together in the presence of one ormore solvents, for example a polar aprotic solvent such as acetonitrileor DMF. In some embodiments, the reactants (formula (III) and formula(VI)) are reacted together at elevated temperature (e.g., at least about50° C., at least about 60° C., at least about 70° C., at least about 80°C., at least about 90° C., at least about 100° C.).

In some embodiments, the reaction mixture also includes a base, forexample a carbonate such as K₂CO₃.

In some embodiments, the reaction mixture also includes a catalyst.

In some embodiments, the compound of formula (VI) is prepared byreacting an amine moiety with an activated acid such as an acidanhydrate or acid halide (e.g., acid chloride) to provide a compound offormula (VI).

The ratio of compounds of formula (III) to formula (VI) can be varied,providing variability in the substitution on the polyamine of formula(III). In general, polyamines having at least about 50% of the hydrogenmoieties substituted with a non-hydrogen moiety are preferred.Accordingly, ratios of compounds of formula (III)/formula (VI) areselected to provide for products having a relatively high degree ofsubstitution of the free amine (e.g., at least about 50%, at least about55%, at least about 60%, at least about 65%, at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 97%, at least about 99%, orsubstantially all). In some preferred embodiments n is 0 in thepolyamine of formula (III), and the ratio of compounds of formula (III)to compounds of formula (VI) is from about 1:3 to about 1:5, preferableabout 1:4. In some preferred embodiments, n is 2 in the polyamine offormula (III), and the ratio of compound of formula (III) to compoundsof formula (VI) is from about 1:3 to about 1:6, preferably about 1:5.

In some embodiments, the compounds of formula (III) and formula (VI) arereacted in a two step process. For example, the first step processincludes a reaction mixture having from about 0.8 about 1.2 molarequivalents of a compound of formula (III), with from about 3.8 to about4.2 molar equivalents of a compound of formula (VI) and the second stepprocess includes addition of about 0.8 to 1.2 molar equivalent ofcompound of formula (VI) to the reaction mixture.

In some embodiments, one or more amine moieties of formula (III) areselectively protected using a protecting group prior to reacting thepolyamine of formula (III) with a compound of formula (IV) or (VI),thereby providing improved selectivity in the synthesis of the finalproduct. For example, one or more primary amines of the polyamine offormula (III) can be protected prior to reaction with a compound offormula (IV) or (VI), providing selectivity for the compound of formula(IV) or (VI) to react with secondary amines. Other protecting groupstrategies can be employed to provide for selectivity towards primaryamines, for example, use of orthogonal protecting groups that can beselectively removed.

Upon completion of the reaction, one or more products having formula (I)can be isolated from the reaction mixture. For example, a compound offormula (I) can be isolated as a single product (e.g., a singlestructural isomer) or as a mixture of product (e.g., a plurality ofstructural isomers and/or a plurality of compounds of formula (I)). Insome embodiments, on or more reaction products can be isolated and/orpurified using chromatography, such as flash chromatography, gravitychromatography (e.g., gravity separation of isomers using silica gel),column chromatography (e.g., normal phase HPLC or RPHPLC), or moving bedchromatography. In some embodiments, a reaction product is purified toprovide a preparation containing at least about 80% of a singlecompound, such as a single structural isomer (e.g., at least about 85%,at least about 90%, at least about 95%, at least about 97%, at leastabout 99%).

In some embodiments, a free amine product is treated with an acid suchas HCl to prove an amine salt of the product (e.g., a hydrochloridesalt). In some embodiments a salt product provides improved properties,e.g., for handling and/or storage, relative to the corresponding freeamine product. In some embodiments, a salt product can prevent or reducethe rate of formation of breakdown product such as N-oxide orN-carbonate formation relative to the corresponding free amine. In someembodiments, a salt product can have improved properties for use in atherapeutic formulation relative to the corresponding free amine.

In some embodiments, a polyamine cationic lipid can be made in using aregioselective synthesis approach. The regioselective synthetic approachprovides a convenient way to make site specific alkylation onnitrogen(s) of the polyamine backbone that leads to synthesis ofspecific alkylated derivatives of interest. In general a compound offormula (I) is initially reacted with a reagent that selectively reactswith primary amines or terminal amines to block them from reacting orinterfering with further reactions and these blockages could beselectively removed at appropriate stages during the synthesis of atarget compound. After blocking terminal amines of a compound of formula(I), one or more of the secondary amines could be selectively blockedwith an orthogonal amine protecting groups by using appropriate molarratios of the reagent and reaction conditions. Selective alkylations,followed by selective deprotection of the blocked amines and furtheralkylation of regenerated amines and appropriate repetition of thesequence of reactions described provides specific compound of interest.For example, terminal amines of triethylenetetramine (1) is selectivelyblocked with primary amine specific protecting groups (e.g.,trifluoroacetamide) under appropriate reaction conditions andsubsequently reacted with excess of orthogonal amine protecting reagent[(Boc)₂O, for e.g.)] in the presence of a base (for e.g.,diisopropylethylamine) to block all internal amines (e.g., Boc).Selective removal of the terminal protecting group and subsequentalkylation of the terminal amines, for instance with an acrylamideprovides a fully terminal amine alkylated derivative of compound 1.Deblocking of the internal amine protection and subsequent alkylationwith calculated amount of an acrylamide for instance yields a partiallyalkylated product 7. Another approach to make compound 7 is to reactterminally protected compound 1 with calculated amount of an orthogonalamine protecting reagent [(Boc)₂O, for e.g.)] to obtain a partiallyprotected derivatives of compound 1. Removal of the terminal amineprotecting groups of partially and selectively protected 1 andsubsequent alkylation of all unprotected amines with an acrylamide, forinstance, yields compound 7 of interest.

Methods of Making Lipids Having a Biocleavable Moiety

In some embodiments, a compound of formula (X) can be made by reacting acompound of formula

with a compound of formula (XII)

wherein R¹, R², and R³ are as defined above.

In some embodiments, the compounds of formulas (XI) and (XII) arereacted in the presence of a coupling agent such as a carbodiimide(e.g., a water soluble carbodiimide such as EDCI).

Other chemical reactions and starting materials can be employed toprovide a compound of formula (X) having two linking groups L¹ and L².For example, the hydroxyl moieties of formula (XI) could be replacedwith amine moieties to provide a precursor to amide or urea linkinggroups.

Upon completion of the reaction, one or more products having formula (X)can be isolated from the reaction mixture. For example, a compound offormula (X) can be isolated as a single product (e.g., a singlestructural isomer) or as a mixture of product (e.g., a plurality ofstructural isomers and/or a plurality of compounds of formula (X)). Insome embodiments, on or more reaction products can be isolated and/orpurified using chromatography, such as flash chromatography, gravitychromatography (e.g., gravity separation of isomers using silica gel),column chromatography (e.g., normal phase HPLC or RPHPLC), or moving bedchromatography. In some embodiments, a reaction product is purified toprovide a preparation containing at least about 80% of a singlecompound, such as a single structural isomer (e.g., at least about 85%,at least about 90%, at least about 95%, at least about 97%, at leastabout 99%).

In some embodiments, a free amine product is treated with an acid suchas HCl to prove an amine salt of the product (e.g., a hydrochloridesalt). In some embodiments a salt product provides improved properties,e.g., for handling and/or storage, relative to the corresponding freeamine product. In some embodiments, a salt product can prevent or reducethe rate of formation of breakdown product such as N-oxide orN-carbonate formation relative to the corresponding free amine. In someembodiments, a salt product can have improved properties for use in atherapeutic formulation relative to the corresponding free amine.

Methods of Making PEG-Lipids

The PEG-lipid compounds can be made, for example, by reacting aglyceride moiety (e.g., a dimyristyl glyceride, dipalmityl glyceride, ordistearyl glyceride) with an activating moiety under appropriateconditions, for example, to provide an activated intermediate that couldbe subsequently reacted with a PEG component having a reactive moietysuch as an amine or a hydroxyl group to obtain a PEG-lipid. For example,a dalkylglyceride (e.g., dimyristyl glyceride) is initially reacted withN,N′-disuccinimidyl carbonate in the presence of a base (for e.g.,triethylamine) and subsequent reaction of the intermediate formed with aPEG-amine (e.g., mPEG2000-NH₂) in the presence of base such as pyridineaffords a PEG-lipid of interest. Under these conditions the PEGcomponent is attached to the lipid moiety via a carbamate linkage. Inanother instance a PEG-lipid can be made, for example, by reacting aglyceride moiety (e.g., dimyristyl glyceride, dipalmityl glyceride,distearyl glyceride, dimyristoyl glyceride, dipalmitoyl glyceride ordistearoyl glyceride) with succinic anhydride and subsequent activationof the carboxyl generated followed by reaction of the activatedintermediate with a PEG component with an amine or a hydroxyl group, forinstance, to obtain a PEG-lipid. In one example, dimyristyl glyceride isreacted with succinic anhydride in the presence of a base such as DMAPto obtain a hemi-succinate. The free carboxyl moiety of thehemi-succinate thus obtained is activated using standard carboxylactivating agents such as HBTU and diisopropylethylamine and subsequentreaction of the activated carboxyl with mPEH2000-NH₂, for instance,yields a PEG-lipid. In this approach the PEG component is linked to thelipid component via a succinate bridge.

Association Complexes

The lipid compounds and lipid preparations described herein can be usedas a component in an association complex, for example a liposome or alipoplex. Such association complexes can be used to administer a nucleicacid based therapy such as an RNA, for example a single stranded ordouble stranded RNA such as dsRNA.

The association complexes disclosed herein can be useful for packagingan oligonucleotide agent capable of modifying gene expression bytargeting and binding to a nucleic acid. An oligonucleotide agent can besingle-stranded or double-stranded, and can include, e.g., a dsRNA, aapre-mRNA, an mRNA, a microRNA (miRNA), a mi-RNA precursor (pre-miRNA),plasmid or DNA, or to a protein. An oligonucleotide agent featured inthe invention can be, e.g., a dsRNA, a microRNA, antisense RNA,antagomir, decoy RNA, DNA, plasmid and aptamer.

Association complexes can include a plurality of components. In someembodiments, an association complex such as a liposome can include anactive ingredient such as a nucleic acid therapeutic (such as anoligonucleotide agent, e.g., dsRNA), a cationic lipid such as a lipiddescribed herein. In some embodiments, the association complex caninclude a plurality of therapeutic agents, for example two or threesingle or double stranded nucleic acid moieties targeting more than onegene or different regions of the same gene. Other components can also beincluded in an association complex, including a PEG-lipid such as aPEG-lipid described herein, or a structural component, such ascholesterol. In some embodiments the association complex also includes afusogenic lipid or component and/or a targeting molecule. In somepreferred embodiments, the association complex is a liposome includingan oligonucleotide agent such as dsRNA, a lipid described herein such asa compound of formula (I) or (X), a PEG-lipid such as a PEG-lipiddescribed herein (e.g., a PEG-lipid of formula (XV), and a structuralcomponent such as cholesterol.

Single Stranded Ribonucleid Acid

Oligonucleotide agents include microRNAs (miRNAs). MicroRNAs are smallnoncoding RNA molecules that are capable of causing post-transcriptionalsilencing of specific genes in cells such as by the inhibition oftranslation or through degradation of the targeted mRNA. An miRNA can becompletely complementary or can have a region of noncomplementarity witha target nucleic acid, consequently resulting in a “bulge” at the regionof non-complementarity. The region of noncomplementarity (the bulge) canbe flanked by regions of sufficient complementarity, preferably completecomplementarity to allow duplex formation. Preferably, the regions ofcomplementarity are at least 8 to 10 nucleotides long (e.g., 8, 9, or 10nucleotides long). A miRNA can inhibit gene expression by repressingtranslation, such as when the microRNA is not completely complementaryto the target nucleic acid, or by causing target RNA degradation, whichis believed to occur only when the miRNA binds its target with perfectcomplementarity. The invention also can include double-strandedprecursors of miRNAs that may or may not form a bulge when bound totheir targets.

In a preferred embodiment an oligonucleotide agent featured in theinvention can target an endogenous miRNA or pre-miRNA. Theoligonucleotide agent featured in the invention can include naturallyoccurring nucleobases, sugars, and covalent internucleoside (backbone)linkages as well as oligonucleotides having non-naturally-occurringportions that function similarly. Such modified or substitutedoligonucleotides are often preferred over native forms because ofdesirable properties such as, for example, enhanced cellular uptake,enhanced affinity for the endogenous miRNA target, and/or increasedstability in the presence of nucleases. An oligonucleotide agentdesigned to bind to a specific endogenous miRNA has substantialcomplementarity, e.g., at least 70, 80, 90, or 100% complementary, withat least 10, 20, or 25 or more bases of the target miRNA.

A miRNA or pre-miRNA can be 18-100 nucleotides in length, and morepreferably from 18-80 nucleotides in length. Mature miRNAs can have alength of 19-30 nucleotides, preferably 21-25 nucleotides, particularly21, 22, 23, 24, or 25 nucleotides. MicroRNA precursors can have a lengthof 70-100 nucleotides and have a hairpin conformation. MicroRNAs can begenerated in vivo from pre-miRNAs by enzymes called Dicer and Droshathat specifically process long pre-miRNA into functional miRNA. ThemicroRNAs or precursor mi-RNAs featured in the invention can besynthesized in vivo by a cell-based system or can be chemicallysynthesized. MicroRNAs can be synthesized to include a modification thatimparts a desired characteristic. For example, the modification canimprove stability, hybridization thermodynamics with a target nucleicacid, targeting to a particular tissue or cell-type, or cellpermeability, e.g., by an endocytosis-dependent or -independentmechanism. Modifications can also increase sequence specificity, andconsequently decrease off-site targeting. Methods of synthesis andchemical modifications are described in greater detail below.

Given a sense strand sequence (e.g., the sequence of a sense strand of acDNA molecule), an miRNA can be designed according to the rules ofWatson and Crick base pairing. The miRNA can be complementary to aportion of an RNA, e.g., a miRNA, a pre-miRNA, a pre-mRNA or an mRNA.For example, the miRNA can be complementary to the coding region ornoncoding region of an mRNA or pre-mRNA, e.g., the region surroundingthe translation start site of a pre-mRNA or mRNA, such as the 5′ UTR. AnmiRNA oligonucleotide can be, for example, from about 12 to 30nucleotides in length, preferably about 15 to 28 nucleotides in length(e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length).

In particular, an miRNA or a pre-miRNA featured in the invention canhave a chemical modification on a nucleotide in an internal (i.e.,non-terminal) region having noncomplementarity with the target nucleicacid. For example, a modified nucleotide can be incorporated into theregion of a miRNA that forms a bulge. The modification can include aligand attached to the miRNA, e.g., by a linker (e.g., see diagrams OT-Ithrough OT-IV below). The modification can, for example, improvepharmacokinetics or stability of a therapeutic miRNA, or improvehybridization properties (e.g., hybridization thermodynamics) of themiRNA to a target nucleic acid. In some embodiments, it is preferredthat the orientation of a modification or ligand incorporated into ortethered to the bulge region of a miRNA is oriented to occupy the spacein the bulge region. For example, the modification can include amodified base or sugar on the nucleic acid strand or a ligand thatfunctions as an intercalator. These are preferably located in the bulge.The intercalator can be an aromatic, e.g., a polycyclic aromatic orheterocyclic aromatic compound. A polycyclic intercalator can havestacking capabilities, and can include systems with 2, 3, or 4 fusedrings. The universal bases described below can be incorporated into themiRNAs. In some embodiments, it is preferred that the orientation of amodification or ligand incorporated into or tethered to the bulge regionof a miRNA is oriented to occupy the space in the bulge region. Thisorientation facilitates the improved hybridization properties or anotherwise desired characteristic of the miRNA.

In one embodiment, an miRNA or a pre-miRNA can include an aminoglycosideligand, which can cause the miRNA to have improved hybridizationproperties or improved sequence specificity. Exemplary aminoglycosidesinclude glycosylated polylysine; galactosylated polylysine; neomycin B;tobramycin; kanamycin A; and acridine conjugates of aminoglycosides,such as Neo-N-acridine, Neo-S-acridine, Neo-C-acridine,Tobra-N-acridine, and KanaA-N-acridine. Use of an acridine analog canincrease sequence specificity. For example, neomycin B has a highaffinity for RNA as compared to DNA, but low sequence-specificity. Anacridine analog, neo-S-acridine has an increased affinity for the HIVRev-response element (RRE). In some embodiments the guanidine analog(the guanidinoglycoside) of an aminoglycoside ligand is tethered to anoligonucleotide agent. In a guanidinoglycoside, the amine group on theamino acid is exchanged for a guanidine group. Attachment of a guanidineanalog can enhance cell permeability of an oligonucleotide agent.

In one embodiment, the ligand can include a cleaving group thatcontributes to target gene inhibition by cleavage of the target nucleicacid. Preferably, the cleaving group is tethered to the miRNA in amanner such that it is positioned in the bulge region, where it canaccess and cleave the target RNA. The cleaving group can be, forexample, a bleomycin (e.g., bleomycin-A₅, bleomycin-A₂, orbleomycin-B₂), pyrene, phenanthroline (e.g., O-phenanthroline), apolyamine, a tripeptide (e.g., lys-tyr-lys tripeptide), or metal ionchelating group. The metal ion chelating group can include, e.g., anLu(III) or EU(III) macrocyclic complex, a Zn(II)2,9-dimethylphenanthroline derivative, a Cu(II) terpyridine, oracridine, which can promote the selective cleavage of target RNA at thesite of the bulge by free metal ions, such as Lu(III). In someembodiments, a peptide ligand can be tethered to a miRNA or a pre-miRNAto promote cleavage of the target RNA, e.g., at the bulge region. Forexample, 1,8-dimethyl-1,3,6,8,10,13-hexaazacyclotetradecane (cyclam) canbe conjugated to a peptide (e.g., by an amino acid derivative) topromote target RNA cleavage. The methods and compositions featured inthe invention include miRNAs that inhibit target gene expression by acleavage or non-cleavage dependent mechanism.

An miRNA or a pre-miRNA can be designed and synthesized to include aregion of noncomplementarity (e.g., a region that is 3, 4, 5, or 6nucleotides long) flanked by regions of sufficient complementarity toform a duplex (e.g., regions that are 7, 8, 9, 10, or 11 nucleotideslong).

For increased nuclease resistance and/or binding affinity to the target,the miRNA sequences can include 2′-O-methyl, 2′-fluorine,2′-O-methoxyethyl, 2′-O-aminopropyl, 2′-amino, and/or phosphorothioatelinkages. Inclusion of locked nucleic acids (LNA), 2-thiopyrimidines(e.g., 2-thio-U), 2-amino-A, G-clamp modifications, and ethylene nucleicacids (ENA), e.g., 2′-4′-ethylene-bridged nucleic acids, can alsoincrease binding affinity to the target. The inclusion of furanosesugars in the oligonucleotide backbone can also decrease endonucleolyticcleavage. An miRNA or a pre-miRNA can be further modified by including a3′ cationic group, or by inverting the nucleoside at the 3′-terminuswith a 3′-3′ linkage. In another alternative, the 3′-terminus can beblocked with an aminoalkyl group, e.g., a 3′ C5-aminoalkyl dT. Other 3′conjugates can inhibit 3′-5′ exonucleolytic cleavage. While not beingbound by theory, a 3′ conjugate, such as naproxen or ibuprofen, mayinhibit exonucleolytic cleavage by sterically blocking the exonucleasefrom binding to the 3′ end of oligonucleotide. Even small alkyl chains,aryl groups, or heterocyclic conjugates or modified sugars (D-ribose,deoxyribose, glucose etc.) can block 3′-5′-exonucleases.

The 5′-terminus can be blocked with an aminoalkyl group, e.g., a5′-O-alkylamino substituent. Other 5′ conjugates can inhibit 5′-3′exonucleolytic cleavage. While not being bound by theory, a 5′conjugate, such as naproxen or ibuprofen, may inhibit exonucleolyticcleavage by sterically blocking the exonuclease from binding to the 5′end of oligonucleotide. Even small alkyl chains, aryl groups, orheterocyclic conjugates or modified sugars (D-ribose, deoxyribose,glucose etc.) can block 3′-5′-exonucleases.

In one embodiment, an miRNA or a pre-miRNA includes a modification thatimproves targeting, e.g. a targeting modification described herein.Examples of modifications that target miRNA molecules to particular celltypes include carbohydrate sugars such as galactose,N-acetylgalactosamine, mannose; vitamins such as folates; other ligandssuch as RGDs and RGD mimics; and small molecules including naproxen,ibuprofen or other known protein-binding molecules.

An miRNA or a pre-miRNA can be constructed using chemical synthesisand/or enzymatic ligation reactions using procedures known in the art.For example, an miRNA or a pre-miRNA can be chemically synthesized usingnaturally occurring nucleotides or variously modified nucleotidesdesigned to increase the biological stability of the molecules or toincrease the physical stability of the duplex formed between the miRNAor a pre-miRNA and target nucleic acids, e.g., phosphorothioatederivatives and acridine substituted nucleotides can be used. Otherappropriate nucleic acid modifications are described herein.Alternatively, the miRNA or pre-miRNA nucleic acid can be producedbiologically using an expression vector into which a nucleic acid hasbeen subcloned in an antisense orientation (i.e., RNA transcribed fromthe inserted nucleic acid will be of an antisense orientation to atarget nucleic acid of interest).

Antisense-Type Oligonucleotide Agents

The single-stranded oligonucleotide agents featured in the inventioninclude antisense nucleic acids. An “antisense” nucleic acid includes anucleotide sequence that is complementary to a “sense” nucleic acidencoding a gene expression product, e.g., complementary to the codingstrand of a double-stranded cDNA molecule or complementary to an RNAsequence, e.g., a pre-mRNA, mRNA, miRNA, or pre-miRNA. Accordingly, anantisense nucleic acid can form hydrogen bonds with a sense nucleic acidtarget.

Given a coding strand sequence (e.g., the sequence of a sense strand ofa cDNA molecule), antisense nucleic acids can be designed according tothe rules of Watson and Crick base pairing. The antisense nucleic acidmolecule can be complementary to a portion of the coding or noncodingregion of an RNA, e.g., a pre-mRNA or mRNA. For example, the antisenseoligonucleotide can be complementary to the region surrounding thetranslation start site of a pre-mRNA or mRNA, e.g., the 5′ UTR. Anantisense oligonucleotide can be, for example, about 10 to 25nucleotides in length (e.g., 11, 12, 13, 14, 15, 16, 18, 19, 20, 21, 22,23, or 24 nucleotides in length). An antisense oligonucleotide can alsobe complementary to a miRNA or pre-miRNA.

An antisense nucleic acid can be constructed using chemical synthesisand/or enzymatic ligation reactions using procedures known in the art.For example, an antisense nucleic acid (e.g., an antisenseoligonucleotide) can be chemically synthesized using naturally occurringnucleotides or variously modified nucleotides designed to increase thebiological stability of the molecules or to increase the physicalstability of the duplex formed between the antisense and target nucleicacids, e.g., phosphorothioate derivatives and acridine substitutednucleotides can be used. Other appropriate nucleic acid modificationsare described herein. Alternatively, the antisense nucleic acid can beproduced biologically using an expression vector into which a nucleicacid has been subcloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest).

An antisense agent can include ribonucleotides only,deoxyribonucleotides only (e.g., oligodeoxynucleotides), or bothdeoxyribonucleotides and ribonucleotides. For example, an antisenseagent consisting only of ribonucleotides can hybridize to acomplementary RNA, and prevent access of the translation machinery tothe target RNA transcript, thereby preventing protein synthesis. Anantisense molecule including only deoxyribonucleotides, ordeoxyribonucleotides and ribonucleotides, e.g., DNA sequence flanked byRNA sequence at the 5′ and 3′ ends of the antisense agent, can hybridizeto a complementary RNA, and the RNA target can be subsequently cleavedby an enzyme, e.g., RNAse H. Degradation of the target RNA preventstranslation. The flanking RNA sequences can include 2′-O-methylatednucleotides, and phosphorothioate linkages, and the internal DNAsequence can include phosphorothioate internucleotide linkages. Theinternal DNA sequence is preferably at least five nucleotides in lengthwhen targeting by RNAseH activity is desired.

For increased nuclease resistance, an antisense agent can be furthermodified by inverting the nucleoside at the 3′-terminus with a 3′-3′linkage. In another alternative, the 3′-terminus can be blocked with anaminoalkyl group.

In one embodiment, an antisense oligonucleotide agent includes amodification that improves targeting, e.g. a targeting modificationdescribed herein.

Decoy-Type Oligonucleotide Agents

An oligonucleotide agent featured in the invention can be a decoynucleic acid, e.g., a decoy RNA. A decoy nucleic acid resembles anatural nucleic acid, but is modified in such a way as to inhibit orinterrupt the activity of the natural nucleic acid. For example, a decoyRNA can mimic the natural binding domain for a ligand. The decoy RNAtherefore competes with natural binding target for the binding of aspecific ligand. The natural binding target can be an endogenous nucleicacid, e.g., a pre-miRNA, miRNA, premRNA, mRNA or DNA. For example, ithas been shown that over-expression of HIV trans-activation response(TAR) RNA can act as a “decoy” and efficiently bind HIV tat protein,thereby preventing it from binding to TAR sequences encoded in the HIVRNA.

In one embodiment, a decoy RNA includes a modification that improvestargeting, e.g. a targeting modification described herein.

The chemical modifications described above for miRNAs and antisenseRNAs, and described elsewhere herein, are also appropriate for use indecoy nucleic acids.

Aptamer-Type Oligonucleotide Agents

An oligonucleotide agent featured in the invention can be an aptamer. Anaptamer binds to a non-nucleic acid ligand, such as a small organicmolecule or protein, e.g., a transcription or translation factor, andsubsequently modifies (e.g., inhibits) activity. An aptamer can foldinto a specific structure that directs the recognition of the targetedbinding site on the non-nucleic acid ligand. An aptamer can contain anyof the modifications described herein.

In one embodiment, an aptamer includes a modification that improvestargeting, e.g. a targeting modification described herein.

The chemical modifications described above for miRNAs and antisenseRNAs, and described elsewhere herein, are also appropriate for use indecoy nucleic acids.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features andadvantages of the invention will be apparent from the description anddrawings, and from the claims.

This application incorporates all cited references, patents, and patentapplications by references in their entirety for all purposes.

In one aspect, the invention features antagomirs. Antagomirs are singlestranded, double stranded, partially double stranded and hairpinstructured chemically modified oligonucleotides that target a microRNA.

An antagomir consisting essentially of or comprising at least 12 or morecontiguous nucleotides substantially complementary to an endogenousmiRNA and more particularly agents that include 12 or more contiguousnucleotides substantially complementary to a target sequence of an miRNAor pre-miRNA nucleotide sequence. Preferably, an antagomir featured inthe invention includes a nucleotide sequence sufficiently complementaryto hybridize to a miRNA target sequence of about 12 to 25 nucleotides,preferably about 15 to 23 nucleotides. More preferably, the targetsequence differs by no more than 1, 2, or 3 nucleotides from a sequenceshown in Table 1, and in one embodiment, the antagomir is an agent shownin Table 2a-e. In one embodiment, the antagomir includes anon-nucleotide moiety, e.g., a cholesterol moiety. The non-nucleotidemoiety can be attached, e.g., to the 3′ or 5′ end of the oligonucleotideagent. In a preferred embodiment, a cholesterol moiety is attached tothe 3′ end of the oligonucleotide agent.

Antagomirs are stabilized against nucleolytic degradation such as by theincorporation of a modification, e.g., a nucleotide modification. Inanother embodiment, the antagomir includes a phosphorothioate at leastthe first, second, or third internucleotide linkage at the 5′ or 3′ endof the nucleotide sequence. In yet another embodiment, the antagomirincludes a 2′-modified nucleotide, e.g., a 2′-deoxy, 2′-deoxy-2′-fluoro,2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl (2′-O-AP),2′-O-dimethylaminoethyl (2′-O-DMAOE), 2′-O-dimethylaminopropyl(2′-O-DMAP), 2′-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or2′-O—N-methylacetamido (2′-O-NMA). In a particularly preferredembodiment, the antagomir includes at least one 2′-O-methyl-modifiednucleotide, and in some embodiments, all of the nucleotides of theantagomir include a 2′-O-methyl modification.

An antagomir that is substantially complementary to a nucleotidesequence of an miRNA can be delivered to a cell or a human to inhibit orreduce the activity of an endogenous miRNA, such as when aberrant orundesired miRNA activity, or insufficient activity of a target mRNA thathybridizes to the endogenous miRNA, is linked to a disease or disorder.In one embodiment, an antagomir featured in the invention has anucleotide sequence that is substantially complementary to miR-122 (seeTable 1), which hybridizes to numerous RNAs, including aldolase A mRNA,N-myc downstram regulated gene (Ndrg3) mRNA, IQ motif containing GTPaseactivating protein-1 (Iqgap1) mRNA, HMG-CoA-reductase (Hmgcr) mRNA, andcitrate synthase mRNA and others. In a preferred embodiment, theantagomir that is substantially complementary to miR-122 isantagomir-122 (Table 2a-e). Aldolase A deficiencies have been found tobe associated with a variety of disorders, including hemolytic anemia,arthrogryposis complex congenita, pituitary ectopia, rhabdomyolysis,hyperkalemia. Humans suffering from aldolase A deficiencies alsoexperience symptoms that include growth and developmental retardation,midfacial hypoplasia, hepatomegaly, as well as myopathic symptoms. Thusa human who has or who is diagnosed as having any of these disorders orsymptoms is a candidate to receive treatment with an antagomir thathybridizes to miR-122.

Double-Stranded Ribonucleic Acid (dsRNA)

In one embodiment, the invention provides a double-stranded ribonucleicacid (dsRNA) molecule packaged in an association complex, such as aliposome, for inhibiting the expression of a gene in a cell or mammal,wherein the dsRNA comprises an antisense strand comprising a region ofcomplementarity which is complementary to at least a part of an mRNAformed in the expression of the gene, and wherein the region ofcomplementarity is less than 30 nucleotides in length, generally 19-24nucleotides in length, and wherein said dsRNA, upon contact with a cellexpressing said gene, inhibits the expression of said gene by at least40%. The dsRNA comprises two RNA strands that are sufficientlycomplementary to hybridize to form a duplex structure. One strand of thedsRNA (the antisense strand) comprises a region of complementarity thatis substantially complementary, and generally fully complementary, to atarget sequence, derived from the sequence of an mRNA formed during theexpression of a gene, the other strand (the sense strand) comprises aregion which is complementary to the antisense strand, such that the twostrands hybridize and form a duplex structure when combined undersuitable conditions. Generally, the duplex structure is between 15 and30, more generally between 18 and 25, yet more generally between 19 and24, and most generally between 19 and 21 base pairs in length.Similarly, the region of complementarity to the target sequence isbetween 15 and 30, more generally between 18 and 25, yet more generallybetween 19 and 24, and most generally between 19 and 21 nucleotides inlength. The dsRNA of the invention may further comprise one or moresingle-stranded nucleotide overhang(s). The dsRNA can be synthesized bystandard methods known in the art as further discussed below, e.g., byuse of an automated DNA synthesizer, such as are commercially availablefrom, for example, Biosearch, Applied Biosystems, Inc.

The dsRNAs suitable for packaging in the association complexes describedherein can include a duplex structure of between 18 and 25 basepairs(e.g., 21 base pairs). In some embodiments, the dsRNAs include at leastone strand that is at least 21 nt long. In other embodiments, the dsRNAsinclude at least one strand that is at least 15, 16, 17, 18, 19, 20, ormore contiguous nucleotides.

The dsRNAs suitable for packaging in the association complexes describedherein can contain one or more mismatches to the target sequence. In apreferred embodiment, the dsRNA contains no more than 3 mismatches. Ifthe antisense strand of the dsRNA contains mismatches to a targetsequence, it is preferable that the area of mismatch not be located inthe center of the region of complementarity. If the antisense strand ofthe dsRNA contains mismatches to the target sequence, it is preferablethat the mismatch be restricted to 5 nucleotides from either end, forexample 5, 4, 3, 2, or 1 nucleotide from either the 5′ or 3′ end of theregion of complementarity.

In one embodiment, at least one end of the dsRNA has a single-strandednucleotide overhang of 1 to 4, generally 1 or 2 nucleotides. Generally,the single-stranded overhang is located at the 3′-terminal end of theantisense strand or, alternatively, at the 3′-terminal end of the sensestrand. The dsRNA may also have a blunt end, generally located at the5′-end of the antisense strand. Such dsRNAs have improved stability andinhibitory activity, thus allowing administration at low dosages, i.e.,less than 5 mg/kg body weight of the recipient per day. Generally, theantisense strand of the dsRNA has a nucleotide overhang at the 3′-end,and the 5′-end is blunt. In another embodiment, one or more of thenucleotides in the overhang is replaced with a nucleoside thiophosphate.

In yet another embodiment, a dsRNA packaged in an association complex,such as a liposome, is chemically modified to enhance stability. Suchnucleic acids may be synthesized and/or modified by methods wellestablished in the art, such as those described in “Current protocols innucleic acid chemistry”, Beaucage, S. L. et al. (Edrs.), John Wiley &Sons, Inc., New York, N.Y., USA, which is hereby incorporated herein byreference. Chemical modifications may include, but are not limited to 2′modifications, modifications at other sites of the sugar or base of anoligonucleotide, introduction of non-natural bases into theoligonucleotide chain, covalent attachment to a ligand or chemicalmoiety, and replacement of internucleotide phosphate linkages withalternate linkages such as thiophosphates. More than one suchmodification may be employed.

Chemical linking of the two separate dsRNA strands may be achieved byany of a variety of well-known techniques, for example by introducingcovalent, ionic or hydrogen bonds; hydrophobic interactions, van derWaals or stacking interactions; by means of metal-ion coordination, orthrough use of purine analogues. Such chemically linked dsRNAs aresuitable for packaging in the association complexes described herein.Generally, the chemical groups that can be used to modify the dsRNAinclude, without limitation, methylene blue; bifunctional groups,generally bis-(2-chloroethyl)amine;N-acetyl-N′-(p-glyoxylbenzoyl)cystamine; 4-thiouracil; and psoralen. Inone embodiment, the linker is a hexa-ethylene glycol linker In thiscase, the dsRNA are produced by solid phase synthesis and thehexa-ethylene glycol linker is incorporated according to standardmethods (e.g., Williams, D. J., and K. B. Hall, Biochem. (1996)35:14665-14670). In a particular embodiment, the 5′-end of the antisensestrand and the 3′-end of the sense strand are chemically linked via ahexaethylene glycol linker. In another embodiment, at least onenucleotide of the dsRNA comprises a phosphorothioate orphosphorodithioate groups. The chemical bond at the ends of the dsRNA isgenerally formed by triple-helix bonds.

In yet another embodiment, the nucleotides at one or both of the twosingle strands may be modified to prevent or inhibit the degradationactivities of cellular enzymes, such as, for example, withoutlimitation, certain nucleases. Techniques for inhibiting the degradationactivity of cellular enzymes against nucleic acids are known in the artincluding, but not limited to, 2′-amino modifications, 2′-amino sugarmodifications, 2′-F sugar modifications, 2′-F modifications, 2′-alkylsugar modifications, 2′-O-alkoxyalkyl modifications like2′-O-methoxyethyl, uncharged and charged backbone modifications,morpholino modifications, 2′-O-methyl modifications, and phosphoramidate(see, e.g., Wagner, Nat. Med. (1995) 1:1116-8). Thus, at least one2′-hydroxyl group of the nucleotides on a dsRNA is replaced by achemical group, generally by a 2′-F or a 2′-O-methyl group. Also, atleast one nucleotide may be modified to form a locked nucleotide. Suchlocked nucleotide contains a methylene bridge that connects the2′-oxygen of ribose with the 4′-carbon of ribose. Oligonucleotidescontaining the locked nucleotide are described in Koshkin, A. A., etal., Tetrahedron (1998), 54: 3607-3630) and Obika, S. et al.,Tetrahedron Lett. (1998), 39: 5401-5404). Introduction of a lockednucleotide into an oligonucleotide improves the affinity forcomplementary sequences and increases the melting temperature by severaldegrees (Braasch, D. A. and D. R. Corey, Chem. Biol. (2001), 8:1-7).

Conjugating a ligand to a dsRNA can enhance its cellular absorption aswell as targeting to a particular tissue or uptake by specific types ofcells such as liver cells. In certain instances, a hydrophobic ligand isconjugated to the dsRNA to facilitate direct permeation of the cellularmembrane and or uptake across the liver cells. Alternatively, the ligandconjugated to the dsRNA is a substrate for receptor-mediatedendocytosis. These approaches have been used to facilitate cellpermeation of antisense oligonucleotides as well as dsRNA agents. Forexample, cholesterol has been conjugated to various antisenseoligonucleotides resulting in compounds that are substantially moreactive compared to their non-conjugated analogs. See M. ManoharanAntisense & Nucleic Acid Drug Development 2002, 12, 103. Otherlipophilic compounds that have been conjugated to oligonucleotidesinclude 1-pyrene butyric acid, 1,3-bis-O-(hexadecyl)glycerol, andmenthol. One example of a ligand for receptor-mediated endocytosis isfolic acid. Folic acid enters the cell by folate-receptor-mediatedendocytosis. dsRNA compounds bearing folic acid would be efficientlytransported into the cell via the folate-receptor-mediated endocytosis.Li and coworkers report that attachment of folic acid to the 3′-terminusof an oligonucleotide resulted in an 8-fold increase in cellular uptakeof the oligonucleotide. Li, S.; Deshmukh, H. M.; Huang, L. Pharm. Res.1998, 15, 1540. Other ligands that have been conjugated tooligonucleotides include polyethylene glycols, carbohydrate clusters,cross-linking agents, porphyrin conjugates, delivery peptides and lipidssuch as cholesterol. Other chemical modifications for siRNAs have beendescribed in Manoharan, M. RNA interference and chemically modifiedsmall interfering RNAs. Current Opinion in Chemical Biology (2004),8(6), 570-579.

In certain instances, conjugation of a cationic ligand tooligonucleotides results in improved resistance to nucleases.Representative examples of cationic ligands are propylammonium anddimethylpropylammonium. Interestingly, antisense oligonucleotides werereported to retain their high binding affinity to mRNA when the cationicligand was dispersed throughout the oligonucleotide. See M. ManoharanAntisense & Nucleic Acid Drug Development 2002, 12, 103 and referencestherein.

The ligand-conjugated dsRNA of the invention may be synthesized by theuse of a dsRNA that bears a pendant reactive functionality, such as thatderived from the attachment of a linking molecule onto the dsRNA. Thisreactive oligonucleotide may be reacted directly withcommercially-available ligands, ligands that are synthesized bearing anyof a variety of protecting groups, or ligands that have a linking moietyattached thereto. The methods of the invention facilitate the synthesisof ligand-conjugated dsRNA by the use of, in some preferred embodiments,nucleoside monomers that have been appropriately conjugated with ligandsand that may further be attached to a solid-support material. Suchligand-nucleoside conjugates, optionally attached to a solid-supportmaterial, are prepared according to some preferred embodiments of themethods of the invention via reaction of a selected serum-binding ligandwith a linking moiety located on the 5′ position of a nucleoside oroligonucleotide. In certain instances, a dsRNA bearing an aralkyl ligandattached to the 3′-terminus of the dsRNA is prepared by first covalentlyattaching a monomer building block to a controlled-pore-glass supportvia a long-chain aminoalkyl group. Then, nucleotides are bonded viastandard solid-phase synthesis techniques to the monomer building-blockbound to the solid support. The monomer building block may be anucleoside or other organic compound that is compatible with solid-phasesynthesis.

The dsRNA used in the conjugates of the invention may be convenientlyand routinely made through the well-known technique of solid-phasesynthesis. Equipment for such synthesis is sold by several vendorsincluding, for example, Applied Biosystems (Foster City, Calif.). Anyother means for such synthesis known in the art may additionally oralternatively be employed. It is also known to use similar techniques toprepare other oligonucleotides, such as the phosphorothioates andalkylated derivatives.

Teachings regarding the synthesis of particular modifiedoligonucleotides may be found in the following U.S. Pat. Nos. 5,138,045and 5,218,105, drawn to polyamine conjugated oligonucleotides; U.S. Pat.No. 5,212,295, drawn to monomers for the preparation of oligonucleotideshaving chiral phosphorus linkages; U.S. Pat. Nos. 5,378,825 and5,541,307, drawn to oligonucleotides having modified backbones; U.S.Pat. No. 5,386,023, drawn to backbone-modified oligonucleotides and thepreparation thereof through reductive coupling; U.S. Pat. No. 5,457,191,drawn to modified nucleobases based on the 3-deazapurine ring system andmethods of synthesis thereof; U.S. Pat. No. 5,459,255, drawn to modifiednucleobases based on N-2 substituted purines; U.S. Pat. No. 5,521,302,drawn to processes for preparing oligonucleotides having chiralphosphorus linkages; U.S. Pat. No. 5,539,082, drawn to peptide nucleicacids; U.S. Pat. No. 5,554,746, drawn to oligonucleotides havingβ-lactam backbones; U.S. Pat. No. 5,571,902, drawn to methods andmaterials for the synthesis of oligonucleotides; U.S. Pat. No.5,578,718, drawn to nucleosides having alkylthio groups, wherein suchgroups may be used as linkers to other moieties attached at any of avariety of positions of the nucleoside; U.S. Pat. Nos. 5,587,361 and5,599,797, drawn to oligonucleotides having phosphorothioate linkages ofhigh chiral purity; U.S. Pat. No. 5,506,351, drawn to processes for thepreparation of 2′-O-alkyl guanosine and related compounds, including2,6-diaminopurine compounds; U.S. Pat. No. 5,587,469, drawn tooligonucleotides having N-2 substituted purines; U.S. Pat. No.5,587,470, drawn to oligonucleotides having 3-deazapurines; U.S. Pat.No. 5,223,168, and U.S. Pat. No. 5,608,046, both drawn to conjugated4′-desmethyl nucleoside analogs; U.S. Pat. Nos. 5,602,240, and5,610,289, drawn to backbone-modified oligonucleotide analogs; U.S. Pat.Nos. 6,262,241, and 5,459,255, drawn to, inter alia, methods ofsynthesizing 21-fluoro-oligonucleotides.

In the ligand-conjugated dsRNA and ligand-molecule bearingsequence-specific linked nucleosides of the invention, theoligonucleotides and oligonucleosides may be assembled on a suitable DNAsynthesizer utilizing standard nucleotide or nucleoside precursors, ornucleotide or nucleoside conjugate precursors that already bear thelinking moiety, ligand-nucleotide or nucleoside-conjugate precursorsthat already bear the ligand molecule, or non-nucleoside ligand-bearingbuilding blocks.

When using nucleotide-conjugate precursors that already bear a linkingmoiety, the synthesis of the sequence-specific linked nucleosides istypically completed, and the ligand molecule is then reacted with thelinking moiety to form the ligand-conjugated oligonucleotide.Oligonucleotide conjugates bearing a variety of molecules such assteroids, vitamins, lipids and reporter molecules, has previously beendescribed (see Manoharan et al., PCT Application WO 93/07883). In apreferred embodiment, the oligonucleotides or linked nucleosides of theinvention are synthesized by an automated synthesizer usingphosphoramidites derived from ligand-nucleoside conjugates in additionto the standard phosphoramidites and non-standard phosphoramidites thatare commercially available and routinely used in oligonucleotidesynthesis.

The dsRNAs packaged in the association complexes described herein caninclude one or more modified nucleosides, e.g., a 2′-O-methyl,2′-O-ethyl, 2′-O-propyl, 2′-O-allyl, 2′-O-aminoalkyl or2′-deoxy-2′-fluoro group in the nucleosides. Such modifications conferenhanced hybridization properties to the oligonucleotide. Further,oligonucleotides containing phosphorothioate backbones have enhancednuclease stability. Thus, functionalized, linked nucleosides can beaugmented to include either or both a phosphorothioate backbone or a2′-O-methyl, 2′-O-ethyl, 2′-O-propyl, 2′-O-aminoalkyl, 2′-O-allyl or2′-deoxy-2′-fluoro group. A summary listing of some of theoligonucleotide modifications known in the art is found at, for example,PCT Publication WO 200370918.

In some embodiments, functionalized nucleoside sequences possessing anamino group at the 5′-terminus are prepared using a DNA synthesizer, andthen reacted with an active ester derivative of a selected ligand.Active ester derivatives are well known to those skilled in the art.Representative active esters include N-hydrosuccinimide esters,tetrafluorophenolic esters, pentafluorophenolic esters andpentachlorophenolic esters. The reaction of the amino group and theactive ester produces an oligonucleotide in which the selected ligand isattached to the 5′-position through a linking group. The amino group atthe 5′-terminus can be prepared utilizing a 5′-Amino-Modifier C6reagent. In one embodiment, ligand molecules may be conjugated tooligonucleotides at the 5′-position by the use of a ligand-nucleosidephosphoramidite wherein the ligand is linked to the 5′-hydroxy groupdirectly or indirectly via a linker. Such ligand-nucleosidephosphoramidites are typically used at the end of an automated synthesisprocedure to provide a ligand-conjugated oligonucleotide bearing theligand at the 5′-terminus.

Examples of modified internucleoside linkages or backbones include, forexample, phosphorothioates, chiral phosphorothioates,phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters,methyl and other alkyl phosphonates including 3′-alkylene phosphonatesand chiral phosphonates, phosphinates, phosphoramidates including3′-amino phosphoramidate and aminoalkylphosphoramidates,thionophosphoramidates, thionoalkylphosphonates,thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′linkages, 2′-5′ linked analogs of these, and those having invertedpolarity wherein the adjacent pairs of nucleoside units are linked 3′-5′to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixed salts and free-acidforms are also included.

Representative United States Patents relating to the preparation of theabove phosphorus-atom-containing linkages include, but are not limitedto, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243;5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717;5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677;5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253;5,571,799; 5,587,361; 5,625,050; and 5,697,248, each of which is hereinincorporated by reference.

Examples of modified internucleoside linkages or backbones that do notinclude a phosphorus atom therein (i.e., oligonucleosides) havebackbones that are formed by short chain alkyl or cycloalkyl intersugarlinkages, mixed heteroatom and alkyl or cycloalkyl intersugar linkages,or one or more short chain heteroatomic or heterocyclic intersugarlinkages. These include those having morpholino linkages (formed in partfrom the sugar portion of a nucleoside); siloxane backbones; sulfide,sulfoxide and sulfone backbones; formacetyl and thioformacetylbackbones; methylene formacetyl and thioformacetyl backbones; alkenecontaining backbones; sulfamate backbones; methyleneimino andmethylenehydrazino backbones; sulfonate and sulfonamide backbones; amidebackbones; and others having mixed N, O, S and CH₂ component parts.

Representative United States patents relating to the preparation of theabove oligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; and 5,677,439, each of which is hereinincorporated by reference.

In certain instances, an oligonucleotide included in an associationcomplex, such as a liposome, may be modified by a non-ligand group. Anumber of non-ligand molecules have been conjugated to oligonucleotidesin order to enhance the activity, cellular distribution or cellularuptake of the oligonucleotide, and procedures for performing suchconjugations are available in the scientific literature. Such non-ligandmoieties have included lipid moieties, such as cholesterol (Letsinger etal., Proc. Natl. Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharanet al., Bioorg. Med. Chem. Lett., 1994, 4:1053), a thioether, e.g.,hexyl-5-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992,660:306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3:2765), athiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533), analiphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaraset al., EMBO J., 1991, 10:111; Kabanov et al., FEBS Lett., 1990,259:327; Svinarchuk et al., Biochimie, 1993, 75:49), a phospholipid,e.g., di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990,18:3777), a polyamine or a polyethylene glycol chain (Manoharan et al.,Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid(Manoharan et al., Tetrahedron Lett., 1995, 36:3651), a palmityl moiety(Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or anoctadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke etal., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative UnitedStates patents that teach the preparation of such oligonucleotideconjugates have been listed above. Typical conjugation protocols involvethe synthesis of oligonucleotides bearing an aminolinker at one or morepositions of the sequence. The amino group is then reacted with themolecule being conjugated using appropriate coupling or activatingreagents. The conjugation reaction may be performed either with theoligonucleotide still bound to the solid support or following cleavageof the oligonucleotide in solution phase. Purification of theoligonucleotide conjugate by HPLC typically affords the pure conjugate.

The modifications described above are appropriate for use with anoligonucleotide agent as described herein.

Fusogenic Lipids

The term “fusogenic” refers to the ability of a lipid or other drugdelivery system to fuse with membranes of a cell. The membranes can beeither the plasma membrane or membranes surrounding organelles, e.g.,endosome, nucleus, etc. Examples of suitable fusogenic lipids include,but are not limited to dioleoylphosphatidylethanolamine (DOPE), DODAC,DODMA, DODAP, or DLinDMA. In some embodiments, the association complexinclude a small molecule such as an imidzole moiety conjugated to alipid, for example, for endosomal release.

PEG or PEG-Lipids

In addition to cationic and fusogenic lipids, the association complexesinclude a bilayer stabilizing component (BSC) such as an ATTA-lipid or aPEG-lipid. Examplary lipids are as follows: PEG coupled todialkyloxypropyls (PEG-DAA) as described in, e.g., WO 05/026372, PEGcoupled to diacylglycerol (PEG-DAG) as described in, e.g., U.S. PatentPublication Nos. 20030077829 and 2005008689), PEG coupled tophosphatidylethanolamine (PE) (PEG-PE), or PEG conjugated to ceramides,or a mixture thereof (see, U.S. Pat. No. 5,885,613). In a preferredembodiment, the association includes a PEG-lipid described here, forexample a PEG-lipid of formula (XV), (XV′) or (XVI). In one preferredembodiment, the BSC is a conjugated lipid that inhibits aggregation ofthe SPLPs. Suitable conjugated lipids include, but are not limited toPEG-lipid conjugates, ATTA-lipid conjugates, cationic-polymer-lipidconjugates (CPLs) or mixtures thereof. In one preferred embodiment, theSPLPs comprise either a PEG-lipid conjugate or an ATTA-lipid conjugatetogether with a CPL.

PEG is a polyethylene glycol, a linear, water-soluble polymer ofethylene PEG repeating units with two terminal hydroxyl groups. PEGs areclassified by their molecular weights; for example, PEG 2000 has anaverage molecular weight of about 2,000 daltons, and PEG 5000 has anaverage molecular weight of about 5,000 daltons. PEGs are commerciallyavailable from Sigma Chemical Co. and other companies and include, forexample, the following: monomethoxypolyethylene glycol (MePEG-OH),monomethoxypolyethylene glycol-succinate (MePEG-S),monomethoxypolyethylene glycol-succinimidyl succinate (MePEG-S—NHS),monomethoxypolyethylene glycol-amine (MePEG-NH.sub.2),monomethoxypolyethylene glycol-tresylate (MePEG-TRES), andmonomethoxypolyethylene glycol-imidazolyl-carbonyl (MePEG-IM). Inaddition, monomethoxypolyethyleneglycol-acetic acid(MePEG-CH.sub.2COOH), is particularly useful for preparing the PEG-lipidconjugates including, e.g., PEG-DAA conjugates.

In a preferred embodiment, the PEG has an average molecular weight offrom about 550 daltons to about 10,000 daltons, more preferably of about750 daltons to about 5,000 daltons, more preferably of about 1,000daltons to about 5,000 daltons, more preferably of about 1,500 daltonsto about 3,000 daltons and, even more preferably, of about 2,000daltons, or about 750 daltons. The PEG can be optionally substituted byan alkyl, alkoxy, acyl or aryl group. PEG can be conjugated directly tothe lipid or may be linked to the lipid via a linker moiety. Any linkermoiety suitable for coupling the PEG to a lipid can be used including,e.g., non-ester containing linker moieties and ester-containing linkermoieties. In a preferred embodiment, the linker moiety is a non-estercontaining linker moiety. As used herein, the term “non-ester containinglinker moiety” refers to a linker moiety that does not contain acarboxylic ester bond (—OC(O)—). Suitable non-ester containing linkermoieties include, but are not limited to, amido (—C(O)NH—), amino(—NR—), carbonyl (—C(O)—), carbamate (—NHC(O)O—), urea (—NHC(O)NH—),disulphide (—S—S—), ether (—0-), succinyl (—(O)CCH.sub.2CH.sub.2C(O)—),succinamidyl (—NHC(O)CH.sub.2CH.sub.2C(O—)NH—), ether, disulphide, etc.as well as combinations thereof (such as a linker containing both acarbamate linker moiety and an amido linker moiety). In a preferredembodiment, a carbamate linker is used to couple the PEG to the lipid.

In other embodiments, an ester containing linker moiety is used tocouple the PEG to the lipid. Suitable ester containing linker moietiesinclude, e.g., carbonate (—OC(O)O—), succinoyl, phosphate esters(—O—(O)POH—O—), sulfonate esters, and combinations thereof.

Targeting Agents

In some embodiments, the association complex includes a targeting agent.For example, a targeting agent can be included in the surface of theassociation complex (e.g., liposome) to help direct the associationcomplex to a targeted area of the body. An example of targeting agentsgalactose, mannose, and folate. Other examples of targeting agentsinclude small molecule receptors, peptides and antibodies. In someembodiments, the targeting agent is conjugated to the therapeutic moietysuch as oligonucleotide agent. In some embodiments, the targeting moietyis attached directly to a lipid component of an association complex. Insome embodiments, the targeting moiety is attached directly to the lipidcomponent via PEG preferably with PEG of average molecular weight 2000amu. In some embodiments, the targeting agent is unconjugated, forexample on the surface of the association complex.

Structural Components

In some embodiments, the association complex includes one or morecomponents that improves the structure of the complex (e.g., liposome).In some embodiments, a therapeutic agents such as dsRNA can be attached(e.g., conjugated) to a lipophilic compound such as cholesterol, therebyproviding a lipophilic anchor to the dsRNA. In some embodimentsconjugation of dsRNA to a lipophilic moiety such as cholesterol canimprove the encapsulation efficiency of the association complex.

Properties of Association Complexes

Association complexes such as liposomes are generally particles withhydrodynamic diameter ranging from about 25 nm to 500 nm. In somepreferred embodiments, the association complexes are less than 500 nm,e.g., from about 25 to about 400 nm, e.g., from about 25 nm to about 300nm, preferably about 120 nm or less.

In some embodiments, the weight ratio of total excipients within theassociation complex to RNA is less than about 20:1, for example about15:1. In some preferred embodiments, the weight ratio is less than 10:1,for example about 7.5:1.

In some embodiments the association complex has a pKa such that theassociation complex is protonated under endozomal conditions (e.g.,facilitating the rupture of the complex), but is not protonated underphysiological conditions.

In some embodiments, the association complex provides improved in vivodelivery of an oligonucleotide such as dsRNA. In vivo delivery of anoligonucleotide can be measured, using a gene silencing assay, forexample an assay measuring the silencing of Factor VII.

In vivo Factor VII Silencing Experiments

C57BL/6 mice received tail vein injections of saline or various lipidformulations. Lipid-formulated siRNAs are administered at varying dosesin an injection volume of 10 μL/g animal body weight. Twenty-four hoursafter administration, serum samples are collected by retroorbital bleed.Serum Factor VII concentrations are determined using a chromogenicdiagnostic kit (Coaset Factor VII Assay Kit, DiaPharma) according tomanufacturer protocols.

Methods of Making Association Complexes

In some embodiments, an association complex is made by contacting atherapeutic agent such as an oligonucleotide with a lipid in thepresence of solvent and a buffer. In some embodiments, a plurality oflipids are included in the solvent, for example, one or more of acationic lipid (e.g., a polyamine containing lipid or a lipid includinga biocleavable moiety as described herein), a PEG-lipid, a targetinglipid or a fusogenic lipid.

In some embodiments, the buffer is of a strength sufficient to protonatesubstantially all amines of an amine containing lipid such as lipiddescribed herein, e.g., a lipid of formula (I) or formula (X).

In some embodiments, the buffer is an acetate buffer, such as sodiumacetate (pH of about 5). In some embodiments, the buffer is present insolution at a concentration of from about 100 mM and about 300 mM.

In some embodiments, the solvent is ethanol. For example, in someembodiments, the mixture includes at least about 90% ethanol, or 100%ethanol.

In some embodiments, the method includes extruding the mixture toprovide association complexes having particles of a size withhydrodynamic diameter less than about 500 nm (e.g., a size from about 25nm to about 300 nm, for example in some preferred embodiments theparticle sizes ranges from about 40-120 nm). In some embodiments, themethod does not include extrusion of the mixture.

In one embodiment, a liposome is prepared by providing a solution of alipid described herein mixed in a solution with cholesterol, PEG,ethanol, and a 25 mM acetate buffer to provide a mixture of about pH 5.The mixture is gently vortexed, and to the mixture is added sucrose. Themixture is then vortexed again until the sucrose is dissolved. To thismixture is added a solution of siRNA in acetate buffer, vortexinglightly for about 20 minutes. The mixture is then extruded (e.g., atleast about 10 times, e.g., 11 times or more) through at least onefilter (e.g., two 200 nm filters) at 40° C., and dialyzed against PBS atpH 7.4 for about 90 minutes at RT.

In one embodiment, a liposome is prepared without extruding the liposomemixture. A lipid described herein is combined with cholesterol, PEG, andsiRNA in 100% ethanol, water, and an acetate buffer having aconcentration from about 100 mM to about 300 mM (pH of about 5). Thecombination is rapidly mixed in 90% ethanol. Upon completion, themixture is dialyzed (or treated with ultrafiltration) against an acetatebuffer having a concentration from about 100 mM to about 300 mM (pH ofabout 5) to remove ethanol, and then dialyzed (or treated withultrafiltration) against PBS to change buffer conditions.

Association complexes can,be formed in the absence of a therapeuticagent such as single or double stranded nucleic acid, and then uponformation be treated with one or more therpauetically active single ordouble stranded nucleic acid moieties to provide a loaded associationcomplex, i.e., an association complex that is loaded with thetherpaueitcally active nucleic acids. The nucleic acid can be entrappedwithin the association complex, adsorbed to the surface of theassociation complex or both. For example, methods of forming associationcomplexes such as liposomes above can be used to form associationcomplexes free of a therapeutic agent, such as a nucleic acid, forexample a single or double stranded RNA such as siRNA. Upon formation ofthe association complex, the complex can then be treated with thetherapeutic agent such as siRNA to provide a loaded association complex.

In one embodiment, a mixture including cationic lipid such as a lipiddescribed in formula (I), preferably a cationic lipid of the followingformula

cholesterol, and a PEG-lipid, for example a PEG-lipid described herein,such as the PEG-lipid below,

are provided in ethanol (e.g., 100% ethanol) and combined with anaqueous buffer such as aqueous NaOAc, to provide unloaded associationcomplexes. The association complexes are then optionally extruded,providing a more uniform size distribution of the association complexes.The association complexes are then treated with the thereapeutic agentsuch as siRNA in ethanol (e.g., 35% ethanol) to thereby provide a loadedassociation complex. In some embodiments, the association complex isthen treated with a process that removes the ethanol, such as dialysis.

Characterization of Association Complexes

Association complexes prepared by any of the methods above arecharacterized in a similar manner Association complexes are firstcharacterized by visual inspection. In general, preferred associationcomplexes are whitish translucent solutions free from aggregates orsediment. Particle size and particle size distribution oflipid-nanoparticles are measured by dynamic light scattering using aMalvern Zetasizer Nano ZS (Malvern, USA). Preferred particles are 20-300nm, more preferrably, 40-100 nm in size. In some preferred embodiments,the particle size distribution is unimodal. The total siRNAconcentration in the formulation, as well as the entrapped fraction, isestimated using a dye exclusion assay. A sample of the formulated siRNAis incubated with the RNA-binding dye Ribogreen (Molecular Probes) inthe presence or absence of a formulation disrupting surfactant, 0.5%Triton-X100. The total siRNA in the formulation is determined by thesignal from the sample containing the surfactant, relative to a standardcurve. The entrapped fraction is determined by subtracting the “free”siRNA content (as measured by the signal in the absence of surfactant)from the total siRNA content. Percent entrapped siRNA is typically >85%.

Methods of Using Association Complexes and Compositions Including theSame

Pharmaceutical Compositions Comprising Oligonucleotide Agents

An oligonucleotide agent assembled in an association complex can beadministered, e.g., to a cell or to a human, in a single-stranded ordouble-stranded configuration. An oligonucleotide agent that is in adouble-stranded configuration is bound to a substantially complementaryoligonucleotide strand. Delivery of an oligonucleotide agent in a doublestranded configuration may confer certain advantages on theoligonucleotide agent, such as an increased resistance to nucleases.

In one embodiment, the invention provides pharmaceutical compositionsincluding an oligonucleotide agent packaged in an association complex,such as a liposome, as described herein, and a pharmaceuticallyacceptable carrier. The pharmaceutical composition comprising thepackaged oligonucleotide agent is useful for treating a disease ordisorder associated with the expression or activity of a target gene,such as a pathological process which can be mediated by down regulatinggene expression. Such pharmaceutical compositions are formulated basedon the mode of delivery. One example is compositions that are formulatedfor delivery to a specific organ/tissue, such as the liver, viaparenteral delivery.

The pharmaceutical compositions featured in the invention areadministered in dosages sufficient to inhibit expression of a targetgene.

In general, a suitable dose of a packaged oligonucleotide agent will besuch that the oligonucleotide agent delivered is in the range of 0.01 to5.0 milligrams per kilogram body weight of the recipient per day,generally in the range of 1 microgram to 1 mg per kilogram body weightper day. The pharmaceutical composition may be administered once daily,or the oligonucleotide agent may be administered as two, three, or moresub-doses at appropriate intervals throughout the day or even usingcontinuous infusion or delivery through a controlled releaseformulation. In that case, the oligonucleotide agent contained in eachsub-dose must be correspondingly smaller in order to achieve the totaldaily dosage. The dosage unit can also be compounded for delivery overseveral days, e.g., using a conventional sustained release formulationwhich provides sustained release of the packaged oligonucleotide agentover a several day period. Sustained release formulations are well knownin the art.

The skilled artisan will appreciate that certain factors may influencethe dosage and timing required to effectively treat a subject, includingbut not limited to the severity of the disease or disorder, previoustreatments, the general health and/or age of the subject, and otherdiseases present. Moreover, treatment of a subject with atherapeutically effective amount of a composition can include a singletreatment or a series of treatments. Estimates of effective dosages andin vivo half-lives for the individual oligonucleotide agents packaged inthe association complexes can be made using conventional methodologiesor on the basis of in vivo testing using an appropriate animal model, asdescribed elsewhere herein.

Advances in mouse genetics have generated a number of mouse models forthe study of various human diseases. Such models are used for in vivotesting of oligonucleotide agents packaged in lipophilic compositions,as well as for determining a therapeutically effective dose.

Any method can be used to administer an oligonucleotide agent packagedin an association complex, such as a liposome, to a mammal. For example,administration can be direct; oral; or parenteral (e.g., bysubcutaneous, intraventricular, intramuscular, or intraperitonealinjection, or by intravenous drip). Administration can be rapid (e.g.,by injection), or can occur over a period of time (e.g., by slowinfusion or administration of slow release formulations).

An oligonucleotide agent packaged in an association complex can beformulated into compositions such as sterile and non-sterile aqueoussolutions, non-aqueous solutions in common solvents such as alcohols, orsolutions in liquid or solid oil bases. Such solutions also can containbuffers, diluents, and other suitable additives. For parenteral,intrathecal, or intraventricular administration, an oligonucleotideagent can be formulated into compositions such as sterile aqueoussolutions, which also can contain buffers, diluents, and other suitableadditives (e.g., penetration enhancers, carrier compounds, and otherpharmaceutically acceptable carriers).

The oligonucleotide agents packaged in an association complex can beformulated in a pharmaceutically acceptable carrier or diluent. A“pharmaceutically acceptable carrier” (also referred to herein as an“excipient”) is a pharmaceutically acceptable solvent, suspending agent,or any other pharmacologically inert vehicle. Pharmaceuticallyacceptable carriers can be liquid or solid, and can be selected with theplanned manner of administration in mind so as to provide for thedesired bulk, consistency, and other pertinent transport and chemicalproperties. Typical pharmaceutically acceptable carriers include, by wayof example and not limitation: water; saline solution; binding agents(e.g., polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers(e.g., lactose and other sugars, gelatin, or calcium sulfate);lubricants (e.g., starch, polyethylene glycol, or sodium acetate);disintegrates (e.g., starch or sodium starch glycolate); and wettingagents (e.g., sodium lauryl sulfate).

EXAMPLES Example 1 Syntheses and Purification of Compounds 3, 4 and 4,5: Alkylation of Triethylenetetramine Under Michael AdditionCondition—Method 1 (Scheme 1)

In a 350 mL pressure bottle N-dodecylacrylamide 1 (84 g, 0.35 mol)[Slee, Deborah H.; Romano, Suzanne J.; Yu, Jinghua; Nguyen, Truc N.;John, Judy K.; Raheja, Neil K.; Axe, Frank U.; Jones, Todd K.; Ripka,William C. Journal of Medicinal Chemistry (2001), 44(13), 2094-2107] wastaken and the solid was melted under argon by gently heating the vessel.To this melt was added triethylenetetramine 2 (10.2 g, 0.07 mol) and themixture was heated at 90° C. for 5 days. Michael addition oftriethylenetetramine 2 to the acrylamide 1 yielded two five and the solesix alkylated products along with minor amounts of low alkylatedproducts under neat reaction condition. The reaction mixture wasanalyzed by TLC using CH₂Cl₂:MeOH:NEt₃ (90:5:5) as the eluent. The TLCshowed the near complete consumption of the starting acrylamide 1. Thereaction mixture was dissolved in dichloromethane (40 mL), loaded on apre-packed column of silica gel and the mixture was separated usingeluent CH₂Cl₂:MeOH:NEt₃ (48:1:1 to 8:1:1). In order to achieve completeseparation, multiple columns using the same conditions were performedand the following pure products were obtained. The required fiveaddition products 3 and 4 were isolated along with the six additionproduct 5. In this reaction mixture some of the lower addition productswere also detected in the TLC and the LC-MS of the crude reactionmixture.

N-Dodecyl-3-((2-dodecylcarbamoyl-ethyl)-{2-[(2-dodecylcarbamoyl-ethyl)-2-{(2-dodecylcarbamoyl-ethyl)-[2-(2-dodecylcarbamoyl-ethylamino)-ethyl]-amino}-ethyl-amino)propionamide.One of the two 5-alkylated derivatives, compound 3 (isomer I), wasisolated as light yellow foam (12 g, 13%). MS m/z 672 (M+2H/2), 448(M+3H/3). ¹H NMR CDCl₃ δ 0.87 (t, J=6.5 Hz, 15H), 1.20-1.39 (m, 92H),1.46-1.57 (m, 12H), 2.20-2.50 (m, 16H), 2.60-2.78 (m, 10H), 3.10-3.25(m, 12H), 6.98 (bs, 3H), 7.41 (bs, 1H), 7.63 (bs, 1H), 8.85 (bs, 1H).¹³C NMR CDCl₃ δ 14.33, 22.90, 27.37, 29.59, 29.67, 29.88, 29.89, 29.92,32.13, 39.74, 172.77.

(3-[(2-{2-[{2-Bis-(2-dodecylcarbamoyl-ethyl)-amino]-ethyl}-(2-dodecylcarbamoyl-ethyl)-amino]-ethylamino}-ethyl)-(2-dodecylcarbamoyl-ethyl)-amino]-N-dodecyl-propionamide).Second 5-alkylated derivative, compound 4 (isomer II) was isolated as awhite powder (13.7 g, 14%). MS m/z 672 (M+2H/2), 448 (M+3H/3). ¹H NMRCDCl₃ δ 0.87 (t, J=6.5 Hz, 15H), 1.20-1.39 (m, 92H), 1.44-1.54 (m, 12H),2.30-2.45 (m, 8H), 2.46-2.54 (m, 8H), 2.55-2.85 (m, 10H), 3.15-3.30 (m,12H), 6.98 (bs, 3H), 7.41 (bs, 1H), 7.63 (bs, 1H), 8.85 (bs, 1H). ¹³CNMR CDCl₃ δ 14.33, 22.89, 27.28, 27.38, 29.59, 29.69, 29.88, 29.89,29.92, 32.13, 39.65, 39.74, 50.84, 172.63, 172.75, 172.81.

Along with this a pure mixture of compounds 3 and 4 (11.6 g, 12%) in 2:3(3:4) ratio was also isolated.

3-[{2-[{2-[Bis-(2-dodecylcarbamoyl-ethyl)-amino]-ethyl}-(2-dodecylcarbamoyl-ethyl)-amino]-ethyl}-(2-dodecylcarbamoyl-ethyl)-amino]-ethyl}-(2-dodecylcarbamoyl-ethyl)-amino]-N-dodecyl-propionamide.The six alkylated product 5 was isolated as a cream powder (16.3 g,17%). MS m/z 792 (M+2H/2), 528 (M+3H/3). ¹H NMR DMSO-d₆ δ 0.87 (t, J=7Hz, 18H), 1.15-1.40 (m, 112H), 1.45-1.53 (m, 12H), 2.20-2.35 (m, 12H),2.37-2.50 (m, 12H), 2.64-2.78 (m, 12H), 3.10-3.25 (m, 12H), 7.26 (bs,4H), 7.64 (bs, 2H). ¹³C NMR CDCl₃ δ 14.32, 22.89, 27.34, 27.38 29.59,29.69, 29.90, 29.92, 32.13, 39.77, 50.85, 172.80.

Example 2 Syntheses and Purification of Compounds 3, 4 and 4: Alkylationof Triethylenetetramine Under Michael Addition Condition—Method 2(Scheme 2)

In another experiment, in order to prevent the polymerization of thestarting acrylamide 1 at high temperature, a radical quencherbenzoquinone was added to the reaction mixture.

In this method a similar reaction to that of Method 1 (Example 1) wasperformed except that, a radical quencher benzoquinone was added to thereaction mixture. In a 150 mL pressure bottle N-dodecylacrylamide 1 (24g, 100 mmol) was taken and to this 15 mg of benzoquinone was added andthe solid acrylamide was melted under argon by gently heating thevessel. To this melt was added triethylenetetramine 2 (2.9 g, 20 mmol)and the mixture was heated at 90° C. for 5 days. The reaction mixturewas analyzed by TLC using CH₂Cl₂:MeOH:NEt₃ (90:5:5) as the eluent. TheTLC showed the near complete consumption of the starting acrylamide 1.The reaction mixture was dissolved in dichloromethane (40 mL) and thedesired products 3, 4 and 5 were isolated as described in Example 1. Inthis case a slight increase in the amount of six addition product wasobserved.

Compound 3: The five addition product, isomer I, was isolated as lightyellow foam (3.4 g, 13%). The analytical and spectral data for thiscompound was identical to that of 3 obtained by Method 1.

Compound 4: The five addition product, isomer II, was isolated as awhite powder (3.9 g, 14%). The analytical and spectral data for thiscompound was identical to that of 4 obtained by Method 1. A pure mixtureof isomers 3 and 4 (1.9 g, 7%) was also isolated.

Compound 5: The six addition product was isolated as a cream powder (6.9g, 26%). The analytical and spectral data for this compound wasidentical to that of 5 obtained by Method 1.

Example 3 Syntheses and Purification of Compounds 3, 4 and 4: Alkylationof Triethylenetetramine Under Michael Addition Condition—Method 3(Scheme 3)

In this method the Michael addition was performed in the presence of apromoter like boric acid (Chaudhuri, Mihir K.; Hussain, Sahid; Kantam,M. Lakshmi; Neelima, B. Tetrahedron Letters (2005), 46(48), 8329-8331.)in order to enhance the rate of the reaction.

In this method a similar reaction to that of Method 1 (Example 1) wasperformed except that, a Michael addition promoter, saturated aqueousboric acid was added to the reaction mixture. In a 150 mL pressurebottle N-dodecyl-acrylamide 1 (24 g, 100 mmol) was melted under argon bygently heating the vessel and to this 3 mL of aqueous boric acid wasadded. To this melt was added triethylenetetramine 2 (2.9 g, 20 mmol)and the mixture was heated at 90° C. for 2 days. The reaction mixturewas analyzed by TLC using CH₂Cl₂:MeOH:NEt₃ (90:5:5) as the eluent. TheTLC showed the near complete consumption of the starting acrylamide 1.The reaction mixture was dissolved in dichloromethane (100 mL) and thesolution was stirred with solid sodium bicarbonate and the organic layerwas filtered and concentrated in a rotory evaporator. This crude productwas purified by column chromatography (silica gel) usingCH₂Cl₂:MeOH:NEt₃ (48:1:1 to 8:1:1). In order to achieve completeseparation, multiple columns using the same conditions were performedand the following pure products were obtained. Under this reactioncondition an increase in yields of compound 4 (isomer II) and sixaddition product 5 were achieved.

Compound 3: The five addition product 3, isomer I, was isolated as lightyellow foam (3.1 g, 11%). The analytical and spectral data for thiscompound was identical to that of 3 obtained by Method 1.

Compound 4: The five addition product 4, isomer II, was isolated as awhite powder (5.7 g, 20%). The analytical and spectral data for thiscompound was identical to that of 4 obtained by Method 1. A pure mixtureof isomers 3 and 4 (2.1 g, 7%) was also isolated.

Compound 5: The six addition product 5 was isolated as a cream powder(7.6 g, 28%). The analytical and spectral data for this compound wasidentical to that of 5 obtained by Method 1.

Example 4 Syntheses and Purification of Compounds 3 and 4: Alkylation ofTriethylenetetramine Under Michael Addition Condition—Method 4 (Scheme4)

In another experiment, in order to minimize the formation of the sixaddition product 5, use of solvent was attempted.

In this method a similar reaction to that of Method 1 (Example 1) andMethod 2 (Example 2) was performed except that, the reactions wereperformed in the presence of solvents at 90° C. with stirring. In a 150mL pressure bottle N-dodecyl-acrylamide 1 (10 g, 41.8 mmol) wasdissolved in 20 mL of either acetonitrile or DMF. To this solution wasadded triethylenetetramine 2 (1 g, 6 8 mmol) and the mixture was heatedat 90° C. for 5 days. The reaction mixture was analyzed by TLC usingCH₂Cl₂:MeOH:NEt₃ (90:5:5) as the eluent. The TLC showed the formation ofonly minor amounts of the required five addition product. The majorproduct in this reaction was a mixture of four addition products alongwith very polar lower addition products.

Example 5 Separation of Unreacted Acrylamide from the Reaction Mixtureand/or the Isolated Products 3, 4 and 5

To remove unreacted acrylamide 1 from the reaction mixture, the reactionmixture is diluted with ethyl acetate or DMF and stirred withpolystyrene or polymer bound thiol (or mercaptan) to capture all theacrylamide. The immobilized thiol was added to the solution and gentlyshaken at ambient temperature and filter off the solid. Michael additionof immobilized thiol to acrylamide capture all unreacted acrylamide.Traces of acrylamide as contaminant after isolation of each desiredisomer could also be completely removed under the same condition. Theisolated product 3 (or 4 or 5) is dissolved in DMF or ethyl acetate andgently shaken with the immobilized acrylamide quencher, filter andevaporation of the filtrate in vacuo affords a pure compound 3 (or 4 or5) free of acrylamide contamination.

Example 6 Separation of Primary and Secondary Amine Contaminant fromCompound 5

After column chromatographic separation of compound 5, to remover tracesof primary and secondary amine contaminants, the compound is dissolvedin ethyl acetate or DMF and stirred with solid bound or immobilizedisothiocyanate at ambient temperature overnight. Filter off the solidand evaporation of the filtrate affords a pure compound 5 free of anyprimary or secondary amine contamination.

Example 7 Separation of Primary Amine Contaminants from Compound 3 and 4

After the completion of the reaction the reaction mixture is treatedwith tetrachlorophthalic anhydride in the presence of triethylamine indichloromethane at room temperature and the solvent is evaporated andthe residue stirred with ethyl acetate and the solid is filtered and thefiltrate is concentrated to get the products which lacks the primaryamine contaminant.

TABLE 1 Methods of synthesizing products 3 and 4 Meth- Temper- Sol-Radical od ature Promoter vent Quencher Remarks 1 90° C. None Neat NoneFormation of 3 and 4 in a combined isolated yield of 39%. The sixaddition product 5 was isolated in 17%. Reaction took six days forcompletion. 2 90° C. None Neat Benzo- Benzoquinone was used quinone toprevent the polymeriza- tion of acrylamide 1. The combined yield of 3and 4 was 34%. However 26% of 5 was also isolated. Reaction time same asMethod 1. 3 90° C. Boric acid Neat None Reaction rate enhanced. Thereaction was com- pleted in two days. The combined yield of 3 and 4 was38%. Additional 28% of 5 was also isolated. 4 80- None DMF None Reactionvery sluggish. 120° C. Only lower addition products formed.

Example 8 Methods of Preparation of the Hydrochloride Salts of theProducts 3, 4 and 5

In order to improve the ease of handling and increase the stability ofthe compounds listed above, they were converted into their correspondinghydrochloride salts 6, 7 and 8.

Hydrochloride of compound 3 (6): The amine 3 (9.4 g) was dissolved in100 mL of hot anhydrous 1,4-dioxane and 100 mL of 4M HCl in dioxane wasadded and the mixture was stirred at room temperature overnight.Nitrogen was bubbled into the reaction mixture for 1 h to remove theexcess HCl and the remaining solution was concentrated to ˜10 mL. Tothis heterogeneous mixture 100 mL of EtOAc:hexanes (1:1) was added andthe precipitated product was filtered, washed with ethyl acetate (50mL), hexanes (100 mL) and the resulting powder was dried under vacuum toget the pure product 6 (9.99 g, 96%) as a cream powder. ¹H NMR CDCl₃ δ0.83 (t, J=6.5 Hz, 15H), 1.20-1.39 (m, 92H), 2.64-2.70 (m, 8H),2.90-3.10 (m, 16H), 3.25-3.45 (m, 12H), 3.46-3.64 (m, 4H), 5.20-6.0 (bs,2H), 8.05-8.15 (m, 5H), 10. (bs, 3H). ¹³C NMR CDCl₃ δ 13.83, 22.04,26.48, 28.69, 28.79, 28.90, 29.04, 31.26, 38.71, 168.38, 168.53.Elemental Analysis Calcd. C₈₁H₁₆₃N₉O₅.4HCl.3H₂O: C, 63.05; H, 11.30; N,8.17; Cl, 9.19. Found: C, 63.13; H, 11.06; N, 8.21; Cl, 9.21.

Compound 7

The amine 4 (13.7 g, 10 2 mmol) was converted to the corresponding HClsalt 7 using a similar procedure used above for 3 to obtain 6. Thetetrahydrochloride salt 7 was isolated as a white powder (14.6, 96%). ¹HNMR CDCl₃ δ 0.82 (t, J=6.5 Hz, 15H), 1.20-1.41 (m, 92H), 2.52-2.72 (m,8H), 2.90-3.10 (m, 16H), 3.25-3.45 (m, 12H), 3.46-3.64 (m, 4H), 5.20-6.0(bs, 2H), 8.05-8.15 (m, 5H), 10. (bs, 3H). ¹³C NMR CDCl₃ δ 8.42, 13.84,22.04, 26.48, 28.69, 28.79, 29.00, 31.26, 45.44, 168.53, 168.60.Elemental Analysis: Calcd: C₈₁H₁₆₃N₉O₅.4HCl.2H₂O: C, 63.79; H, 11.30; N,8.17; Cl, 9.34. Found: C, 63.78; H, 11.04; N, 8.40; Cl, 9.73.

Compound 8

The amine 5 (13.7 g, 1.2 mmol) was converted to the corresponding HCl 8using a procedure similar to that described above for the salt 6. Thetetrahydrochloride salt 8 was isolated as a white powder (1.3 g, 96%).¹H NMR DMSO-d₆ δ 0.87 (t, J=7 Hz, 18H), 1.13-1.30 (m, 112H), 1.35-1.53(m, 12H), 2.10-2.25 (m, 12H), 2.30-2.40 (m, 12H), 2.60-2.76 (m, 12H),3.10-3.25 (m, 12H), 7.26 (bs, 4H), 7.64 (bs, 2H), 10.1 (bs, 4H).

Example 9 Selective Protection of Amino Groups on Triethylenetetraminefor Directed Synthesis of Compounds 3 and 4

Step 1: Preparation of compound 10: Triethylenetetramine, 2 (20.55 g,140.52 mmol, purchased from Sigma-Aldrich) in acetonitrile (500 mL) wascooled over an ice bath under constant stirring. Ethyl trifluoroacetate(35.20 mL, 295.09 mmol) was added to the stirring solution and stirredfor 20 h. Solvent and volatiles were removed under reduced pressure anddried under high vacuum to get 9 as white solid (44.4 g, 94%). Theproduct thus obtained could be used for the next reaction withoutfurther purification (Wender P. A. et al. Organic Letters, 2005 7,4815).

Crude compound 9 (23.70, 70 mmol) was dissolved in acetonitrile (400 mL)and stirred over an ice bath. N-(Benzyloxycarbonyloxy) succinate (Z—OSu,43.73 g, 175 mmol, purchased from Novabiochem) and triethylamine (23.40mL, 210 mmol) were added to the reaction mixture and stirred overnight.Solvents were removed and the residue was extracted into dichloromethane(DCM), washed successively with water (two times) and brine, dried overanhydrous sodium sulfate. Solvent was removed in vacuo and residue thusobtained was purified by silica gel column chromatography (gradientelution, 30-70% EtOAc/Hexanes) to obtain compound 10 as white solid(38.2 g, 89%). ¹H NMR (DMSO-d6, 400 MHz) δ=9.60-9.50(m, 2H),7.40-7.20(m, 10H), 5.02(s, 4H), 3.40-3.20(m, 12H). MS: C₂₆H₂₈F₆N₄O₆ Cal.606.19. Found. 607.2 (M⁺).

Step 2: Preparation of compound 11: Compound 10 (12.60 g, 20.78 mmol)was suspended in methanol (MeOH, 150 mL) at ambient temperature and 8Msolution of methylamine in ethanol (40 ml) was added to the suspensionunder constant stirring. All the solids went into solution, afterstirring for 1 h at ambient temperature, the mixture was warmed to 50°C. and stirred for 8 h. Reaction was monitored by TLC. All the solventswere removed under reduced pressure and the residue was purified bysilica gel column chromatography (gradient elution, 10% MeOH/DCM to10:10:80, MeOH:TEA:DCM) to yield the product 11 (7.80 g, 91%) as paleyellow gummy liquid. ¹H NMR (DMSO-d6, 400 MHz) δ=7.80-7.40(m, 10H),5.02-4.94(m, 4H), 3.45-3.05(m, 8H), 2.70-2.55(m, 4H), 2.20(bs, 4H). MS:C₂₂H₃₀N₄O₄ Cal. 414.23. Found 415.20 (M⁺)

Step 3: Preparation of compound 13: Compound 12 was prepared fromtriethylenetetramine, 100 (10.25 g, 70.09 mmol) as described in step 1for the synthesis of compound 9 by reacting with 1.1 molar equivalent ofethyl trifluoroacetate (8.80 mL, 77.10 mmol). Crude 12 thus obtained wasdissolved in anhydrous DCM (400 ml) and cooled to 0° C. (Boc)₂O (53.53mmol, 245.31 mmol) and triethylamine (48 ml, 350 mmol) were added andreaction mixture was allowed to stir overnight. Progress of the reactionwas monitored by TLC. Solvents were removed in vacuo and the residue wasextracted into DCM, washed with water, brine and dried. DCM was removedand the residue was purified by silica gel chromatography (gradientelution 50% EtOAc/Hexane to EtOAc) to obtain the desired product 13(34.20 g, 92%) as white solid. ¹H NMR (DMSO-d6, 400 MHz) δ=9.51-9.38(m,1H), 6.82(bs, 1H), 3.30-3.00(m, 12H), 1.58-1.30(s, 27H). MS:C₂₃H₄₁F₃N₄O₇ Cal. 542.29. Found 543.4(M⁺).

Step 4: Preparation of 14: A solution of compound 13 (25 g, 47.32 mmol)in MeOH (200 mL) was stirred with K₂CO₃ (50 g) in the presence of water(1 mL) at 50° C. overnight. Progress of the reaction was monitored byTLC. Solid K₂CO₃ was filtered off, washed with MeOH, combined washingand solvents were removed in vacuo. Residue obtained was purified bysilica gel column chromatography to yield the desired product 14 (10.2g, 50%) as white solid. ¹H NMR (DMSO-d6, 400 MHz) δ=6.83 (bs, 1H),2.95-3.30 (m, 12H), 2.62-2.50(m, 2H), 1.25-1.45(m, 27H). MS: C₂₁H₄₂N₄O₆Cal. 446.31. Found 447.4(M⁺).

Step 5: Preparation of compound 15: Compound 9 (23.0 g, 68.02 mmol) wasdissolved in a mixture of acetonitrile/dichloromethane (1:1, 300 mL) andcooled to 0° C. Z—OSu (17.00 g, 69 mmol) was added to the solution andstirred for 10 minutes. Triethylamine (23.40 mL, 210 mmol) wassubsequently added to the reaction mixture and allowed to stirovernight. Solvents and triethylamine were removed in vacuo and theresidue was extracted into DCM, washed with water (two times), brine anddried. After removing solvent, the residue was purified by silica gelcolumn chromatography (eluted initially with 20-60% EtOAc/Hexane, thenwith 5% MeOH/DCM) to obtain the desired product 15 (13.3 g) as whitesolid along with side product 10 (8.5 g). ¹H NMR (DMSO-d6, 400 MHz)δ=9.60 (bs, 1H), 9.30(bs, 1H), 7.40-7.28(m, 5H), 5.01(s, 2H),3-40-3.10(m, 8H), 2.70-2.50(m, 4H). MS: C₁₈H₂₂F₆N₄O₄ Cal. 472.15. Found473.1(M⁺).

Step 6: Preparation of compound 16: Treatment of compound 15 (13.4 g,28.38 mmol) with methylamine (50 ml, 8M solution in EtOH) as describedin step 2 yielded a colorless liquid compound 16 (6.10 g, 79%). Theproduct thus obtained could be used for next reaction without furtherpurification. ¹H NMR (DMSO-d6, 400 MHz) δ=7.45-7.20(m, 6H), 5.07(s, 2H),3.45-2.90(m, 8H), 2.60-2.30(m, 4H). MS: C₁₄H₂₄N₄O₂ Cal. 280.19 Found281.2(M⁺).

Example 10 Synthesis of 5-Alkylated Single Isomer 4-Method 1

Step 1: Reaction of 11 with N-dodecylacrylamide: Diamine 11 (1.00 g,2.41 mmol) and N-dodecylacrylamide (3.47 g, 14.50 mmol) were takentogether in a pressure tube and heated at 90° C. for 5 days. Thereaction was monitored by TLC. Once the reaction is over, the mixture isdissolved in dichloromethane and purified by flash chromatography to getthe products 17, 18 and 19.

Step 2: Preparation of compound 20: Compound 19 (2.00 g, 1.46 mmol) isdissolved in a mixture of ethylacetate and methanol (1:2, 15 ml) to that2 eq. of acetic acid is added. The mixture is hydrogenated underpressure (50 psi) using palladium/carbon (0.200 g, 10% wt) as a catalystto get the desired product 20.

Step 3: Preparation of single isomer 4: Compound 20 (1.50 g, 1.36 mmol)and the acrylamide 1 (0.325 mmol, 1.36 mmol) is dissolved in toluene (4mL) and heated at 90° C. days to form compound 4. Progress of thereaction is monitored by TLC. After completion of reaction, the mixtureis cooled to room temperature, dissolved in DCM and purified by flashsilica gel column chromatography to obtain the desired product 4.

Example 11 Synthesis of 5-Alkylated Single Isomer 4-Method 2

Step 1: Preparation of compound 21: Compound 16 (1.0 g, 3.56 mmol) andN-dodecylacrylamide (6.00 g, 7 eq) are taken together in a pressure tubeand heated to obtain compound 21. Progress of the reaction is monitoredby TLC. After completion of the reaction the mixture is dissolved in DCMand purified by flash silica gel chromatography to afford the desiredcompound 21.

Step 2: Preparation of compound 4 from 21: Compound 21 (2.00 g, 1.35mmol) is dissolved in a mixture of ethyl acetate and methanol (1:2, 15ml) to that 2 eq. of acetic acid is added. The mixture is hydrogenatedunder pressure (50 psi) over palladium-carbon (0.200 g, 10% wt) toafford the desired single isomer 4.

Example 12

Synthesis of 5-Alkylated Single Isomer 3-Method 1

Step 1: Preparation of compound 22: Compound 14 (5.06 g, 11.30 mmol) andN-dodecylacrylamide (2.94 g, 12.43 mmol) were taken in toluene andheated at 90° C. for five days. TLC was checked and showed the formationof product. The reaction mixture was directly loaded on a pre-packedcolumn of column silica gel and purified by flash chromatography (5%MeOH/DCM) to afford compound 22 (4.82 g, 62%). ¹H NMR (DMSO-d6, 400 MHz)δ=8.17(bs, 1H), 6.60(bs, 1H), 3.30-2.95(m, 12H), 2.70(t, J=5.80 Hz, 2H),2.60(t, J=6.00 Hz, 2H), 2.18(t, J=6.40 Hz, 2H), 1.35(m, 29H),1.26-1.15(m, 18H), 0.83(t, J=6.00 Hz, 3H). MS: C₃₆H₇₁N₅O₇ Cal. 685.54.Found 686.5(M⁺).

Step 2: Preparation of compound 23: Compound 22 (4.75 g, 6.92 mmol) wasdissolved in dichloromethane (100 mL) and cooled to 0° C. Z—OSu (2.59 g,1.5 eq) was added to the solution and stirred for 10 minutes. Thereaction mixture was subsequently stirred with triethylamine (2.82 mL,20.76 mmol) overnight. Solvent and triethylamine were removed in vacuoand the residue was extracted into dichloromethane, washed successivelywith water (two times) and brine, and dried over anhydrous sodiumsulfate. After removing solvent the residue was purified by flash silicagel column chromatography (5-10% MeOH/DCM) to obtain the desiredcompound 23 (5.33 g, 94%). ¹H NMR (CDCl₃, 400 MHz) δ=7.49-7.25(m, 5H),5.11(s, 2H), 3.60-3.02 (m, 14H), 2.45-45(m, 4H), 1.50-1.35(m, 27H),1.24-1.20(m, 18H), 0.87(t, J=6.00 Hz, 3H). MS: C₄₄H₇₇N₅O₉ Cal. 819.57.Found 820.7(M).

Step 3: Preparation of compound 24: 4M HCl in dioxane (50 mL) was addedinto a solution of compound 23 (5.30 g, 6.50 mmol) in dioxane (100 ml).The reaction mixture was then allowed to stir overnight. Product wasprecipitated out during the course of the reaction. Solvent and HCl wereremoved under vacuum to yield a white solid. The residue was taken inMeOH containing excess triethylamine and the suspension was stirred for1 h to obtain a homogeneous solution. Solvents were removed in vacuo andthe residue was triturated with EtOAc, filtered off the triethylaminehydrochloride salt. Combined filtrate was evaporated under vacuum toobtain a gummy liquid 24 (3.30 g, 98%). ¹H NMR (CDCl₃, 400MHz)δ=7.37-7.28(m, 5H), 5.05(s, 2H), 3.60-3.20(m, 4H), 3.10-2.70(m, 10H),2.40-2.20(m, 4H), 1.40-1.30(m, 2H), 1.25-1.17(m, 18H), 0.81(t, J=6.00Hz, 3H). MS: C₂₉H₅₃N₅O₃ Cal. 519.41. Found 520.4(M⁺).

Step 4: Preparation of compound 25: Compound 24 (1.00 g, 1.925 mmol) andN-dodecylacrylamide (3.70 g, 8 eq) are taken together in a pressure tubeand heated at elevated temperature to form desired compound 25.Formation of the product is monitored by TLC and is subsequentlypurified by flash silica gel column chromatography to afford a purecompound 25.

Step 5: Preparation of compound 3: Compound 25 (2.00 g, 1.35 mmol) isdissolved in a mixture of ethyl acetate and methanol (1:2, 15 ml) tothat 2 eq. of acetic acid is added. The mixture is hydrogenated underpressure (50 psi) over palladium-carbon (0.200 g, 10% wt) to afford thedesired product 3.

Example 13 Synthesis of 5-Alkylated Single Isomer 3-Method 2

Step 1: Preparation of compound 26: Benzyl bromide (1.25 ml, 1.5 eq) toa suspension of compound 22 (4.80 g, 7.00 mmol) and K₂CO₃ (9.67 g, 10eq) in DMF (100 mL) and the mixture was stirred overnight. Progress ofthe reaction was monitored by TLC. Solids were filtered off, washed withMeOH and ethyl acetate. Combined filtrate was concentrated under reducedpressure and the residue thus obtained was purified by silica gel columnchromatography (50-100% EtOAc/Hexane) to afford the desired compound 26(3.30 g, 61%). ¹H NMR (DMSO-d6, 400 MHz) δ=7.77(bs, 2H), 7.28-7.23(m,5H), 6.85-6.70(m, 1H), 3.59(s, 2H), 3.20-2.20(m, 18H), 1.35(s, 27H),1.30-1.23(m, 2H), 1.20-1.15(m, 18H), 6.81(t, J=6.00 Hz, 3H). MS:C₄₃H₇₇N₅O₇ Cal. 775.58. Found 776.5(M⁺)

Step 2: Preparation of compound 27: Compound 26 (3.30 g, 4.25 mmol) indioxane (50 ml) was stirred with 4M HCl (50 mL) in dioxane overnight.Formation of white precipitate was seen during the course of thereaction. Solvent and acid were removed under vacuum and white residuethus obtained was redissolved in methanol containing excesstriethylamine. The homogeneous solution was then evaporated underreduced pressure to obtain while residue. The residue was trituratedwith EtOAc and filtered off triethylamine hydrochloride salt. Filtratewas evaporated under vacuum to afford the desired compound 27 (2.36 g,99%) as gummy liquid. ¹H NMR (CDCl₃, 400 MHz) δ=8.05(t, J=5.5 Hz, 1H),7.40-7.20(m, 5H), 3.58(s, 2H), 3.10-2.30(m, 18H), 1.40-1.30(m, 2H),1.25-1.15(m, 18H), 0.82(t, J=6.00 Hz, 3H). MS: C₂₈H₅₃N₅O Cal. 475.43.Found. 498.4(M+Na)

Step 3: Preparation of compound 28: Neat compound 27 (1.00 g, 2.10 mmol)and N-dodecylacrylamide (4.0 g, 8 eq) are mixed in a pressure tube andheated to elevated temperature to form compound 28. Formation of 28 ismonitored by TLC and LC-MS. After completion of the reaction the productis isolated by chromatographic purification to afford pure compound 28.

Step 4: Preparation of compound 3 from compound 28: Compound 28 (2.00 g,1.40 mmol) is dissolved in a mixture of ethyl acetate and methanol (1:2,15 ml) to that 6 eq. of acetic acid is added. The mixture ishydrogenated under pressure (50 psi) over palladium-carbon (0.200 g, 10%wt) to obtain compound 3

Example 14 Convergent Synthesis of Isomer 3—Method 1

Step 1: Preparation of compounds 30, 31 and 32: Ethylenediamine 29(0.978 ml, 14.63 mmol), N-dodecylacrylamide (7.00 g, 29.26 mmol) andboric acid (100 mg) were taken in 5 mL of water and heated at 90° C. forfour days. Complete disappearance of acrylamide was ascertained by TLCanalysis. The reaction mixture was dissolved in DCM, washed with waterand bicarbonate and dried over sodium sulfate. DCM was removed and theresidue was purified by silica gel column chromatography (2:2:96 to10:10:80% MeOH/TEA/DCM) to get compounds 30 (1.86 g) ¹H NMR (CDCl₃, 400MHz) δ=7.05(bs, 2H), 3.21 (q, J=6.30 Hz, 4H), 2.87(t, J=6.00 Hz, 4H),2.73(s, 4H), 2.34(t, J=6.00 Hz, 4H), 1.57(bs, 2H), 1.49-1.45(m, 4H),1.28-1.19(m, 40H), 0.87(t, J=6.8 Hz, 6H) MS: C₃₂H₆₆N₄O₂ Cal. 538.52.Found 539.50(M⁺). 31 (3.50 g) ¹H NMR (DMSO-d6, 400 MHz) δ=8.20(bs, 1H),3.20-2.15 (m, 22H), 1.36-1.30(m, 6H), 1.25-1.15(m, 30H), 0.81(t, J=6.00Hz, 9H), MS: C₄₇H₉₅N₅O₃ Cal. 777.74. Found 778.7(M⁺) and 32 (1.75 g) ¹HNMR (DMSO-d6, 400 MHz) δ=3.23-2.15 (m, 28H), 1.35-1.45 (m, 8H),1.26-1.15 (m, 40H), 0.82 (t, J=6.00 Hz, 12H). MS: C₆₂H₁₂₄N₆O₄ Cal.1016.97. Found 1018.0 (M⁺).

Step 2: Preparation of compound 33: Compound 31 (1.55 g, 2 mmol) andK₂CO₃ (2.76 g, 20 mmol) are taken in DMF. To that chloroacetaldehydedimethyl acetal (0.453 ml, 4.00 mmol) is added and stirred for 24 h.Reaction is monitored by TLC, filtered off K₂CO₃ washed with MeOH.Solvents are removed under reduced pressure and the residue is subjectedto chromatographic purification to afford compound 33.

Step 3: Preparation of compound 34: Compound 33 (2.00 g, 2.31 mmol) istaken in a mixture of MeOH and DCM, to that PTSA (2.0 eq) is added andreaction mixture is stirred overnight. The solution is neutralized withsodium bicarbonate solution and extract with DCM and dried. Compound ispurified by chromatographic separation to afford the desired product 34.

Step 4: Preparation of single isomer 3 from 34: Compound 34 (2.00 g,2.43 mmol) and 30 (1.31 g, 2.43 mmol) are taken in DCM; to thatactivated molecular sieves is added and stirred for 3 h. The reaction ismonitored by TLC. Once the reaction is over solvents is removed. Theresidue is dissolved in THF and sodium triacetoxyborohydride (5 eq.) andacetic acid are added and stirred overnight. Solvents are removed andextracts with DCM, chromatographic separation of the residue affordspure isomer 3.

Example 15 Convergent Synthesis of Isomer 3—Method 2

The desired single isomer 3 is also prepared from compound 30 byselective protection of one of the nitrogen to obtain compound 35.Compound 35 is subsequently reacted with aldehyde 34 under reductiveconditions to obtain compound 36. Acid treatment of 36 affords desiredcompound 3.

Example 16 Convergent Synthesis of Isomer 3—Method 3

The desired single isomer 3 is also prepared from monobenzylethylenediamine 37. Alkylation of 37 with 1 affords a mixture ofcompounds 38, 39 and 40. Compound 40 is reacted with aldehyde 34 underreductive conditions to obtain compound 41. Hydrogenolysis of 41 affordsthe desired compound 3.

Example 17 Convergent Synthesis of Isomer 4—Method 1

Step 1: Preparation of compounds 43: In a 150 mL pressure bottleN-dodecyl-acrylamide 1 (16.4 g, 68 8 mmol) was melted under argon bygently heating the vessel and to this 3 mL of aqueous boric acid wasadded. To this melt was added Boc protected ethylenediamine 42 (5 g,31.2 mmol) and the mixture was heated at 90° C. overnight. The reactionmixture was analyzed by TLC using CH₂Cl₂:MeOH:NEt₃ (90:5:5) as theeluent. The TLC showed the near complete consumption of the startingacrylamide 1. The reaction mixture was dissolved in dichloromethane (100mL) and the solution was stirred with solid sodium bicarbonate and theorganic layer was filtered and concentrated in a rotory evaporator. Thiscrude product was purified by column chromatography (silica gel) usingCH₂Cl₂:MeOH:NEt₃ (48:1:1 to 8:1:1). The major product in this reactionis the double addition product 43. Minor amounts of mono adduct was alsoobserved.

Step 2: Preparation of compound 44: Compound 43 (2.00 g, 3.13 mmol) istaken in dioxane (50 mL) to that HCl (20 mL, 4M solution in dioxane) isadded and stirred overnight. Solvent is removed to get the compound 44.

Step 3: Preparation of single isomer 4 from 34 and 44: Compound 34 (2.00g, 2.43 mmol) and 44 (1.31 g, 2.43 mmol) are taken in DCM; to thatactivated molecular sieves is added and stirred for 3 h. The reaction ismonitored by TLC. Once the reaction is over solvents are removed. Theresidue is dissolved in THF and sodium triacetoxy borohydride (5 eq.)and acetic acid are added and stirred overnight. Solvents are removedand extracts with DCM, chromatographic separation of the residue affordspure isomer 4.

Example 18 Addition of N-dodecylacrylamide to 1,3-diaminopropane andSubsequent Reduction of the Amide to Amine

In order to study the effect of number of charges in the cationic lipidthe Michael adducts of acrylamide 1 with 1,3-diaminopropane 45 wasinvestigated.

Step 1: Synthesis of 46, 47 and 48: In a 150 mL pressure bottleN-dodecyl-acrylamide 1 (15.4 g, 64 mmol) was melted under argon bygently heating the vessel and to this 3 mL of aqueous boric acid wasadded. To this melt was added 1,3-diaminopropane 44 (1.58 g, 21 mmol)and the mixture was heated at 90° C. overnight. The reaction mixture wasanalyzed by TLC using CH₂Cl₂:MeOH:NEt₃ (90:5:5) as the eluent. The TLCshowed the near complete consumption of the starting acrylamide 1. Thereaction mixture was dissolved in dichloromethane (100 mL) and thesolution was stirred with solid sodium bicarbonate and the organic layerwas filtered and concentrated in a rotory evaporator. This crude productwas purified by column chromatography (silica gel) usingCH₂Cl₂:MeOH:NEt₃ (48:1:1 to 8:1:1). The major product in this reactionis the triple addition product 46. Minor amounts of tetra adduct 47 andbis adduct 48 were also isolated.

N-Dodecyl-3-{(2-dodecylcarbamoyl-ethyl)-[3-(2-dodecylcarbamoyl-ethylamino)-propyl]-amino}-propionamide46. The three addition product 46 was isolated as a white powder (5.7 g,35%). MS m/z 793 (MH⁺). ¹H NMR CDCl₃ δ 0.87 (t, J=6.6 Hz, 9H), 1.20-1.30(m, 60H), 1.42-1.66 (m, 6H), 2.33 (t, J=6 Hz, 4H), 2.38-2.46 (m, 4H),2.60-2.70 (m, 4H), 2.84 (t, 2H), 3.15-3.28 (m, 6H), 6.65 (bs, 1H), 6.99(bs, 3H).

4-[{3-[Bis-(2-dodecylcarbamoyl-ethyl)-amino]-propyl}-(2-dedecylcarbamoyl-ethyl)amino]-N-dodecyl-butyramide47. The four addition product 47 was also isolated in minor amounts.

N-Dodecyl-3-[-(2-dodecylcarbamoyl-ethylamino)-propylamino]-propionamide48. The diadduct 48 was isolated as a cream powder (1.6 g, 10%). MS m/z553 (MH⁺). ¹H NMR CDCl₃ δ 0.89 (t, J=6.6 Hz, 6H), 1.10-1.20 (m, 40H),1.42-1.66 (m, 4H), 2.20 (t, J=6 Hz, 4H), 2.55 (t, 4H), 2.60 (t, 4H),3.00 (m, 4H), 8.00 (bs, 2H).

Step 2: Conversion of Amines 4, 35 and 36 to their CorrespondingHydrochloride Salts 49, 50 and 51.

The amine 46 (5.5 g) was converted to the corresponding HCl 49 using aprocedure similar to the described in Example 8 and the dihydrochloridesalt 49 was isolated as a white powder (5.73 g, 92%). ¹H NMR DMSO-d₆ 80.88 (t, J=7 Hz, 9H), 1.17-1.30 (m, 66H), 1.35-1.45 (m, 6H), 2.10-2.25(m, 2H), 2.55-2.70 (m, 6H), 2.95-3.15 (m, 10H), 3.20-3.35 (m, 6H), 8.16(t, 1H), 8.24 (t, 1H), 9.15 (bs, 1H), 10.65 (bs, 1H).

In a similar procedure to that described in Example 8 the amine 47 istreated with 4M HCl to obtain the dihydrochloride salt 50.

In a similar procedure to that described in Example 8 the amine 48 istreated with 4M HCl to obtain the dihydrochloride salt 51.

Step 3: Reduction of amides 46, 47 and 48 to amines 52, 53 and 54: Amine46 is refluxed in THF with excess of diborane overnight and subsequenttreatment with 4M HCl affords hydrochloride salt of polyamine 52.

A similar treatment of amines 47 and 48 affords the correspondingreduced product 53 and 54 as their respective hydrochloride salt.

Example 19 Reduction of Polyamides 3, 4 and 5 to the CorrespondingPolyamine Dendrimers

Compound 3 is refluxed with large excess of diborane in THF to obtainthe corresponding reduced product 55. After completion of the reaction,the reaction mixture is treated with 4M HCl prior to work-up and theproduct is isolated as its hydrochloride salt. Hydrochloride salts of 56and 57 are also obtained from the corresponding precursors 4 and 5respectively.

Example 20 Polyamino Alkyl Lipids—Reduction of Amides to Amines

Preparation of polyamines 60 from 32: Compound 32 (1.02 g, 1 mmol) istaken in THF (20 ml), to that BH₃.THF (60 ml, 1M in THF) is added andrefluxed for two days. Reaction is monitored by TLC. Removal of THFgives a white residue, which is treated with 1M HCl and extracts intoDCM. Chromatographic separation of the crude products yields purecompound 60.

Preparation of polyamines 58 and 59 from 30 and 31: Reduction of amides30 and 31 under similar conditions described for the preparation 60respectively affords 58 and 59.

Example 21 Synthesis of Polyamido-Polyamino Alkyls—Alkylation of AminesUsing Alkyl Halides

Step 1: preparation of compound 62: A solution of chloroacetyl chloride(10.31 mL, 129.37 mmol) in DCM (200 mL) was cooled over an ice bath andto this a solution of dodecylamine (61, 20.00 g, 107.81 mmol) indichloromethane containing TEA (36.70 ml, 269.5 mmol) was added dropwiseover a period of 1 hr. The reaction mixture tuned brownish-black by thistime, continued the stirring for another hour at 0° C. The reactionmixture was filtered through a sintered funnel, washed with EtOAc,diluted with chloroform, washed successively with water, sodiumbicarbonate solution, 1M HCl and brine. Organic layer was dried oversodium sulfate. Solvents were removed and the residue was purified bysilica gel column chromatography (5-50% EtOAc/Hexane) to afford compound62 (26.00 g, 92%) as brown solid. ¹H NMR (CDCl₃, 400 MHz) δ=6.59(bs,1H), 4.03(s, 2H), 3.25 (q, J=6.00 Hz, 2H), 1.54-1.49(m, 2H),1.45-1.15(m, 18H), 0.86(t, J=6.00 Hz, 3H). MS: C₁₄H₂₈ClNO Cal. 261.19.Found 262.20(M⁺).

Step 2: Preparation of 63, 64 and 65: Triethylenetetramine 2 (1.00 g,6.83 mmol) and chloroacetamide 62 (10.00 g, 5.5 eq) are taken togetherin a mixture of CH₃CN/DMF (1:3), to that K₂CO₃ (9.43 g, 10 eq) and KI(50 mg) are added and heated at 85° C. for three days. The reactionmixture is filtered to remove solids, wash with DCM, solvents areremoved in vacuo and chromatographic separation of the crude residueaffords pure compounds 63, 64 and 65.

Example 22 Synthesis of Polyamido-Polyamino Alkyls—Alkylation of AminesUsing Alkyl Halides with Branched Aminoalkyls

Step 1: Preparation of 67: Chloroacetyl chloride (4.05 mL, 51 mmol) wastaken in DCM (100 mL) and cooled down to 0° C. To this a dichloromethanesolution of N,N-didodecylamine (66, 15.00 g, 42.41 mmol) and TEA (14.43ml, 2.5 eq.) were added dropwise over a period of 1 hr. The reactionmixture tuned brownish-black by this time, after the addition thereaction mixture was stirred for 24 h at ambinet temperature. Thereaction mixture was filtered through a sintered funnel, washed withEtOAc, diluted with chloroform, washed successively with water, sodiumbicarbonate solution, 1M HCl and brine. Organic layer was dried oversodium sulfate. Solvents were removed in vacuo and the residue waspurified by silica gel column chromatography (5-50% EtOAc/Hexane toobtian the required product 67 (12.5 g, 69%) as brownish liquid. 1H NMR(CDCl₃, 400 MHz) δ=4.04(s, 2H), 3.30(m, 4H), 1.50-1.45(m, 2H),1.40-1.20(m, 18H), 0.87(t, J=6.00 Hz, 3H). MS: C₂₆H₅₂ClNO Cal. 430.15.Found 431.2(M⁺).

Step 2: Preparation of 68, 69 and 70: Triethylenetetramine 2 (0.500 g,6.83 mmol) and chloroacetamide 67 (8.10 g, 5.5 eq) are taken together ina mixture of CH₃CN/DMF (1:3), to that K₂CO₃ (4.72 g, 10 eq) and KI (30mg) are added and heated at 85° C. for three days. The reaction mixturewas filtered to remove insoluble solids, wash with DCM, solvents areremoved and chromatographic separation of the residue affords t 68, 69and 70.

Example 23 Addition of N,N-dialkylacrylamide to Polyamines

In order to study the effect of adding more hydrophobic chains to thecationic lipids, didodecylamine was used as a precursor to theacrylamide.

Step 1: Synthesis of N,N-Didodecylacrylamide 71

To a solution of didodecylamine 66 (25 g, 70 7 mmol) anddiisopropylethylamine (18 g, 141 mmol) in anhydrous CH₂Cl₂ (700 mL) at−10° C., a solution of acryloyl chloride (7.68 g, 85 mmol) in CH₂Cl₂(100 mL) was added dropwise over a period of 20 min. After thecompletion of the addition the reaction mixture was stirred for 4 h at0° C. after which the TLC of the reaction mixture showed the completionof the reaction. The reaction mixture was washed with satd. NaHCO₃solution (200 mL), water (200 mL), brine (100 mL) and dried over NaSO₄.Concentration of the organic layer provided the product 71 (28.4 g,100%) which was used as such in the next step. ¹H NMR CDCl₃ δ 0.94 (t,J=6.5 Hz, 6H), 1.05-1.69 (m, 40H), 3.15-3.60 (dt, 4H), 5.64 (d, 1H),6.36 (d, 1H), 6.63 (m, 1H).

Step 2: Reaction of triethyelentetramine 2 and 71

The acrylamide 71 is treated with the amine 2 and after usual work-upand column purification the Michael addition products 72, 73 and 74 areisolated.

Step 3: Synthesis of hydrochloride salts 75, 76 and 77: Each singlecompound obtained is taken in dioxane and 4M HCl in dioxane is added tothe solution and stirred as described in example 8 to yield thecorresponding hydrochloride salt.

Example 24 Alkenylation of Polyamines Using Mono Unsaturated N-AlkylAcrylamide Under Michael Addition Condition

In order to study the effect of double bond in the alkyl chainoleylamine was used as a precursor to the acrylamide 79.

Step 1: Synthesis of compound 79: To a solution of oleylamine 78 (26.75g, 100 mmol) and triethylamine (20 g, 200 mmol) in anhydrous CH₂Cl₂ (200mL) at −10° C., a solution of acryloyl chloride (9.9 g, 110 mmol) inCH₂Cl₂ (100 mL) was added dropwise over a period of 20 min. After thecompletion of the addition the reaction mixture was stirred for 4 h at0° C. after which the TLC of the reaction mixture showed the completionof the reaction. The reaction mixture was washed with satd. NaHCO₃solution (200 mL), water (200 mL), brine (100 mL) and dried over NaSO₄.Concentration of the organic layer provided the product 79 (32 g, 100%)which was used as such in the next step. ¹H NMR CDCl₃ δ 0.91 (t, J=6.5Hz, 3H), 1.05-1.35 (m, 24H), 1.42 (t, 2H), 1.96 (m, 4H), 5.31 (t, 1H),5.33-5.36 (m, 1H), 5.54 (dd, 1H), 6.02 (dd, 1H), 6.18 (dd, 1H), 8.03(bs, 1H).

Step 2: Reaction of Compound 79 with Triethylenetetramine

The acrylamide 79 is treated with triethylenetetramine 2 and after usualwork-up and column purification of the Michael addition products affordspure compounds 80, 81 and 82.

Step 3: Synthesis of hydrochloride salts 83, 84 and 85: Each singlecompound (80, 81 or 82) obtained is taken in dioxane and 4M HCl indioxane is added to the solution and stirred as described in example 8to yield the corresponding hydrochloride salt.

Example 25 Alkenylation of Diamines Using Mono Unsaturated N-AlkylAcrylamide Under Michael Addition Condition

In a similar procedure to that of Example 24 the acrylamide 79 istreated with the diamine 45 and after usual work-up and columnpurification the Michael addition products 86, 87 and 88 are isolated.Treatment of the free amine thus obtained with HCl in dioxane affordsthe corresponding hydrochloride salts 89, 90 and 91 respectively.

Example 26 Alkenylation of Polyamines Using Poly Unsaturated N-AlkylAcrylamide Under Michael Addition Condition

In order to study the effect of polyunsaturation in the alkyl chainlinoleylamine 92 was used as a precursor to the acrylamide 93.

Step 1: Compound 93: Linolylamine 92 is treated with acryloyl chloridein a similar procedure to that of Example 24, step 1 and thecorresponding acrylamide 93 is isolated.

Step 2: Reaction of Compound 93 with Triethylenetetramine

The acrylamide 93 is treated with triethylenetetramine 2 in the presenceof boric acid as described in Example 3 and after usual work-up andcolumn purification of the Michael addition products affords purecompounds 94, 95 and 96.

Step 3: Synthesis of hydrochloride salts 97, 98 and 99: Each singlecompound (94, 95 or 96) obtained is taken in dioxane and 4M HCl indioxane is added to the solution and stirred as described in example 8to yield the corresponding hydrochloride salt.

Example 27 Alkenylation of Diamines Using Poly Unsaturated N-AlkylAcrylamide Under Michael Addition Condition

In a similar procedure to that of Example 3 the acrylamide 93 is treatedwith the diamine 45 in the presence of boric acid and after usualwork-up and column purification the Michael addition products 100, 101and 102 are isolated. Treatment of the free amine thus obtained with HClin dioxane affords the corresponding hydrochloride salts 103, 104 and105 respectively.

Example 28 Alkenylation of Polymines Using Alkyl Acrylates Under MichaelAddition Condition

Method 1: n-Dodecylacrylate (106) is stirred with triethylenetetramine 2in methanol-water at 40° C. to obtain compounds 107, 108 and 109. Theproducts are isolated by chromatographic separation.

Method 2: n-Dodecylacrylate (106) is stirred with triethylenetetramine 2in the presence of boric acid in methanol-water at 40° C. to obtaincompounds 107, 108 and 109. The products are isolated by chromatographicseparation.

Example 29 Alkenylation of Diamines Using Alkyl Acrylates Under MichaelAddition Condition

Method 1: n-Dodecylacrylate (106) is stirred with triethylenetetramine 2in methanol-water at 40° C. to obtain compounds 110, 111 and 112. Theproducts are isolated by chromatographic separation.

Method 2: n-Dodecylacrylate (106) is stirred with triethylenetetramine 2in the presence of boric acid in methanol-water at 40° C. to obtaincompounds 110, 111 and 112. The products are isolated by chromatographicseparation.

Example 30 Synthesis of Octadeca-9,12-dienoic acid3-dimethylamino-2-octadeca-9,12-dienoyloxy-propyl ester 3

To a solution of the linoleic acid (25 g, 89.1 mmol) in anhydrous DMF(60 mL), diisopropyl ethylamine (17 mL, 100 mml) was added at roomtemperature with stirring followed by 3-(dimethylamino)-1,2-propanediol(4.8 g, 40.5 mmol) and EDCI (17.25 g, 89.9 mmol) and the mixture wasstirred at room temperature overnight. The TLC of the reaction mixture(eluent 20% EtOAc in hexanes) showed the completion of the reaction. Thereaction mixture was poured into ice water and extracted with ethylacetate (2×100 mL). The combined organic layers were washed with water(100 mL), saturated NaHCO₃ (100 mL) and dried over Na₂SO₄. Concentrationof the organic layer provided the crude product which was purified bycolumn chromatography (silica gel, eluent: 20% EtOAc in hexanes). Thefractions containing pure product was pooled and concentrated. The pureester was isolated as a clear liquid (5.7 g, 22%). MS m/z 645 (M+H). ¹HNMR CDCl₃ δ 0.88 (t, J=6.3 Hz, 6H), 1.20-1.39 (m, 28H), 1.61 (t, J=4.9Hz, 12H), 2.03-2.08 (m, 8H), 2.26-2.38 (m, 10H), 2.44-2.56 (m, 2H), 2.76(t, J=6.3 Hz, 4H), 4.09 (dd, J=6.1 Hz & 11.9 Hz, 1H), 4.36 (dd, J=3.3 &11.9 Hz, 1H), 5.29-5.34 (m, 1H), 5.34-5.41 (m, 8H). ¹³C NMR CDCl₃ δ14.30, 22.79, 25.08, 25.10, 25.83, 27.40, 29.26, 29.30, 29.34, 29.42,29.55, 29.83, 31.73, 34.32, 34.58, 46.01, 59.37, 64.02, 128.08, 128.24,130.21, 130.42, 173.39, 173.65.

Example 31 Exemplary Procedure for Making a Liposome Using Extrusion

Prepare stock solutions of ND98 (120 mg/ml), cholesterol (25 mg/ml), andC16-PEG-Cer-2000 (100 mg/ml) in 100% ethanol. Store at −20° C. Warm in37° C. water bath prior to preparing formulations (up to 30 minutes ishelpful—it takes a while for the cholesterol to dissolve completely).

2×2 ml Prep

To a 15 ml Falcon tube, add:

1) 125 ul of lipid

2) 200 ul of cholesterol

3) 70 ul of PEG

4) 5 ul of 100% ethanol

5) 600 ul of 25 mM sodium acetate pH 5

6) Mix gently (setting 5) on a vortex

7) Add 20 mg sucrose

8) Vortex again until sucrose has dissolved

9) Add 1 ml of a freshly-prepared (in a new Falcon tube) 1 mg/mlsolution of siRNA in 25 mM sodium acetate (=100 ul of 10 mg/ml siRNA+900ul of 25 mM sodium acetate)

10) Vortex lightly (setting 1, with Falcon tube holder adapter) for 20minutes

11) After 15 minutes (5 minutes remaining), clean extruder

12) Extrude 11 times through two 200 nm filters at 40° C.

13) Dialyze against PBS, pH 7.4 for 90 minutes at RT in 3,500 MWCOPierce cassettes

Example 32 Exemplary Procedure for Making a Liposome without UsingExtrusion

Prepare stock solutions of ND98 (120 mg/ml), cholesterol (25 mg/ml), andC16-PEG-Cer-2000 (100 mg/ml) in 100% ethanol. Store at −20° C. Warm in37° C. water bath prior to preparing formulations (up to 30 minutes ishelpful—it takes a while for the cholesterol to dissolve completely).

To a 15 ml Falcon tube, add:

1) 125 ul of lipid

2) 200 ul of cholesterol

3) 70 ul of PEG

4) 495 ul of 100% ethanol

5) 100 ul of water

6) Prepare 1 ml of 1 mg/ml siRNA in 100-300 mM sodium acetate, pH ˜5

7)Rapidly mix lipids in 90% ethanol with siRNA in acetate buffer

8) Dialyze (or use ultrafiltration) against 100-300 mM sodium acetate,pH ˜5 to remove ethanol

9) Dialyze (or use ultrafiltration) against PBS to change bufferconditions

Example 33 Exemplary Protocol for Quantification of RNA in a LiposomeSample

The procedure below can be used to quantify (1) the proportion ofentrapped siRNA and (2) the total amount of siRNA in a liposome.

Materials:

RiboGreen (Molecular Probes)

2% Triton X-100

TE buffer

Protocol (96-Well Plate Format)

-   1. Dilute samples to be tested in TE buffer such that siRNA    concentration is ˜2 ug/mL (0.4-4 ug/mL). Note dilution of samples.-   2. Array 50 uL of each sample into 2 wells (e.g. samples arrayed    into 2 rows of microplate)-   3. Add 50 uL of TE buffer to one of each of the 2 samples (e.g. top    row samples). This sample will be used to determine “free” siRNA.-   4. Add 50 uL of 2% Triton X-100 to the remaining of the 2 samples    (e.g. bottom row samples). This sample will be used to determine    “total” siRNA.-   5. Prepare standard siRNA dilutions by using known amounts of the    siRNA to be quantified. Start with 50 uL of 4 ug/mL, and do 2-fold    dilutions. Add 50 uL of 2% Triton X-100 to each of the standard    sample dilutions.-   6. Incubate for 15 min at room temperature.-   7. Add 100 uL of diluted RiboGreen to all of the samples. Diluted    RiboGreen to be used at 1:100 dilution.-   8. Read plate in fluorimeter (Victor2) using FITC settings.

Calculations:

Final volume in wells will be 200 uL.

RiboGreen will be at 1:200 final dilution.

Triton X-100 will be at 0.5%.

Standards will be dilutions starting from 1 ug/mL.

Plot Standard Curve, perform linear fit.

Determine Entrapment %=100*(1-“free” signal/“total” signal)

Determine [siRNA]: First convert “total” signal to concentration usingthe standard curve, then multiply by dilution factor.

Example 34 Comparison of Lipid Moieties as Formulated into Liposomes

The effectiveness of lipid compositions can be tested by determining therelative ability of a lipid to deliver an siRNA moiety to a target. Forexample, the silencing of a target indicates that the siRNA is deliveredinto the cell. Applicants have compared liposome complexes that includeeach of the following lipid moieties together with siRNA that is used tosilence Factor VII (FVII).

Initially unpurified reaction mixtures were used. Different ND98reaction mixtures were generated by synthesizing product at differentND:98 monomer ratios: ND:98=1:1, 2:1, 3:1, 4:1, 5:1, and 6:1. ND98 isgenerated by reacting ND, the structure of which is provided below:

with amine 98, the structure of which is provided below

in the ratios provided above (i.e., ND:98=1:1, 2:1, 3:1, 4:1, 5:1, and6:1).

Liposomes were formulated atND98:cholesterol:FED2000-CerC16:siRNA=15:0.8:7:1 (wt ratios). Liposomesprepared with ND:98=1:1 and 2:1 precipitated drying formulation and werenot characterized further.

Table 1, below provides the average particle size and percent entrapmentof the liposomes using the various monomer ratios (i.e, the numberindicating the ratio of ND relative to 98).

TABLE 1 Z-Avg. Particle size (nm) % Entrapment ND98 3 56 >95 ND98 456 >95 ND98 5 81 93 ND98 6 72 74FIG. 1 provides the results of the FVII silencing assay for the variousmonomer ratios using an experimental dosing of 2 mg/kg siRNA. Theresults suggest that the ND98 5 tail moiety and/or ND 98 6 tail moietyare the active species as these are the most abundants species on theND98 6:1 preparation. As described a 5 tail moiety indicates a compoundwhere 5 of the hydrogens on the starting amine 98 have been reacted witha starting acrylamide moiety ND. A 6 tail moiety indicates a compoundwhere 6 of the hydrogens on the starting amine 98 have been reacted withan acrylamide moiety ND. Accordingly, the number of “tails” indicatesthe number of reacted hydrogens on the starting amine.

Example 35 Determination of Preferred Lipid Isomer

Applicants purified ND98 lipid products. ND98 lipid moieties are thelipid moieties resulting in the reaction of ND, the structure of whichis provided below:

with amine 98, the structure of which is provided below

Applicants tested 4-tail mixed isomers of ND98 (i.e., where four of theamine hydrogens have been reacted with the ND acrylamide above), singlestructural isomers of 5-tail ND98 (i.e., where for of the aminehydrogens have been reacted with the ND acrylamide above). Examples ofthe two 5 tail isomers are provided below:

Liposomes of the purified ND98 products were formulated with thefollowing components in the following ratios:ND98:cholesterol:PEG2000-CerC16:siRNA=15:5:7:1 (wt ratios).

Table 2, below provides the average particle size and percent entrapmentof the liposomes using the various monomer ratios (i.e, the numberindicating the ratio of ND relative to 98).

TABLE 2 Z-Avg. Particle size (nm) % Entrapment ND98 1 88 >95 ND98 2 10486 ND98 3 115 86 ND98 4 92 >95 For the purposes of table 2 and FIG. 2:ND98 1 = 5-tailed (isomer I); ND98 2 = 5-tailed (isomer I + II); ND98 3= 5-tailed (isomer II); and ND98 4 = 4-tailed.

The liposomes where administered with siRNA at a does of 2.5 mg/kg, andevaluated for the silencing of FVII. FIG. 2 provides the results of the4 tailed isomer mixture, the single 5 tailed isomers (i.e., isomer I andII) and the mixture of 5 tailed isomers (i.e., isomer I and II).

Example 36 Determination of Preferred ND98 Isomer

A purified isomer of 6 tailed ND98 was prepared and purified. ND98structure corresponds with those described in examples 34 and 35 above.The 6 tail indicates that all of the hydrogens of amine 98 have beenreacted with the ND starting material. With this lipid startingmaterial, liposomes were formulated at the following ratios:ND98:cholesterol:PEG2000-CerC16:siRNA=15:5:7:1 (wt ratios). FIG. 3demonstrates the effectiveness of the ND98 6 tail isomer in delivery ofsiRNA, which effectively silenced FVII.

Example 37 Liposome Particle Size Using Various ND98 Lipid StartingMaterials

A plurality of lipid starting materials having the ND98 structures (asprovided in examples 34 and 35 above) were formulated into liposomes.The particle size of the liposomes were evaluated, the results of whichare provided in table 3 below:

Formulation Particle Diameter (nm) ND98 3 (Exp 1) 56 ND98 4 (Exp 1) 56ND98 5 (Exp 1) 81 ND98 6 (Exp 1) 72 ND98 1 (Exp 2) 88 ND98 2 (Exp 2) 104ND98 3 (Exp 2) 115 ND98 4 (Exp 2) 92 6-tailed ND98 (Exp 3) 127

Example 38 Extrusion Free Liposome Formulation

Liposome complexes were prepared using ND98 lipids. The formulationsinclude the following ratios:ND98:cholesterol:PEG2000-CerC16:siRNA=15:5:7:1 (wt. ratios). Theliposomes were prepared without extrusion, as generally described inExample 32 above. Two samples were prepared, a first sample having thefollowing: 100 mM=siRNA prepared in 100 mM sodium acetate with a firstdialysis step in 100 mM acetate; and a second sample having 300 mM=siRNAprepared in 300 mM sodium acetate with a first dialysis step in 300 mMacetate.

FIG. 4 shows the results of an FVII silencing assay, demonstrating thecomparative activity of the formulations made using the variousprocesses.

Example 39 Regioselective Synthesis of Cationic Lipid 7—Strategy 1

Step 1. Preparation of compound 9: Triethylenetetramine, 1 (48.83 g,0.334 mol, purchased from Sigma-Aldrich) in anhydrous acetonitrile (500mL) was cooled over an ice bath under constant stirring. Ethyltrifluoroacetate (79.6 mL, 0.668 mol) was added to the solution andafter completion of the addition the reaction mixture was allowed towarm to room temperature and stirred for 20 h. Solvent and volatileswere removed under reduced pressure and the residue was dissolved inminimum amount of warm dichloromethane (100 mL) and to it cold hexaneswas added with stirring. The precipitated product was cooled in ice andfiltered to get a white solid (112.2 g, 99%).

Step 2. Synthesis of(2-{tert-butoxycarbonyl-[2-(2,2,2-trifluoro-acetylamino)ethyl]-amino}-2-(2,2,2-trifluoro-acetylamino)ethyl]-carbamicacid tert-butyl ester 113

The trifluoroacetamide 9 (112.2 g, 0.332 mol) was dissolved inCH₂Cl₂/THF (600 mL/100 mL) and to it diisopropylethylamine (129.25 g, 1mol) was added and stirred over an ice bath. Di-tert-butyl dicarbonate(145 g, 0.664 mol, purchased from Sigma Aldrich) in CH₂Cl₂ (100 mL) wasadded drop wise to the reaction mixture and stirred overnight. Solventswere removed and the residue was stirred with a saturated solution ofNaHCO₃ (400 mL) and filtered and washed with hexanes (100 mL) and driedin vacuo at 45° C. overnight to obtain the pure diboc compound as awhite solid (167 g, 94%). ¹H NMR for 113 (DMSO-d6, 400 MHz)δ=9.60-9.40(m, 2H), 3.35-3.15(m, 12H), 1.36(s, 18H) MS: C₁₅H₂₄F₆N₄O₄Cal. 438.17. Found 439.20 (M⁺) MS: C₂₀H₃₂F₆N₄O₆ Cal. 538.22. Found539.20(M⁺).

Step 3. Synthesis of(2-amino-ethyl)-{2-[(2-amino-ethyl)-tert-butoxycarbonyl-amino]-ethyl}carbamicacid tert-butyl ester

The acetamide 113 (167 g, 0.31 mol) was taken in a stainless steelpressure reactor and to it a solution of methylamine (33% by wt) inethanol (200 ml) was added. The mixture was warmed to 90° C. and stirredfor 24 h. Reaction was monitored by mass spectra. All the solvents wereremoved under reduced pressure and the residue was subjected to highvacuum at 80° C. to yield the product 114 (103 g, 96%) as gummy liquidand this compound could be used for the next reaction with out furtherpurification. ¹H NMR (CDCl₃, 400 MHz) δ=3.20-3.00(m, 4H), 2.62-2.38(m,8H), 1.32(s, 9H). MS: C₁₁H₂₆N₄O₂ Cal. 246.21. Found 246.20(M⁺).

Step 4. Synthesis of Michael addition product 115

The diamine 114 (103 g, 0.297 mmol), N-dodecylacrylamide (356 g, 1.487mol) and saturated solution of boric acid in water (30 mL) were takentogether in a pressure reactor and heated at 90° C. for 4 days. Thereaction was monitored by TLC and Mass spectra. The reaction mixture wasextracted into dichloromethane (DCM), washed successively with NaHCO₃solution and brine, dried over anhydrous sodium sulfate. Solvent wasremoved in vacuo and residue thus obtained was purified by silica gelcolumn chromatography (gradient elution-Ethyl acetate then 3-10%MeOH/DCM) to obtain 115 as a pale yellow solid (228 g, 59%). MS:C₇₆H₁₅₀N₈O₈ Cal. 1303.16. Found 1304.20 (M⁺).

Step 5. Preparation of diamine-116

4M HCl in dioxane (500 mL) was added to a solution of the diboc compound115 (228 g, 0.175 mol) in methanol (100 mL) and the mixture was stirredat room temperature for 2 days. The reaction was monitored by Massspectra. After the complete disappearance of the starting diboccompound, the precipitated hydrochloride salt was filtered, washed withTHF (100 mL) and dried to get the pure salt as a white powder (178 g,93%). The above salt was treated with saturated NaHCO₃ (1 L) andextracted with dichloromethane (3×600 mL). The combined organic extractswere dried and concentrated to isolate the tetramer as a white solid(164 g, 85%). MS: C₆₆H₁₃₄N₈O₄ Cal. 1103.05. Found 1104.10 (M⁺).

Step 6. Synthesis of 117: Compound 116 (164 g, 149 mmol),N-dodecylacrylamide (35.6 g, 149 mmol) and saturated solution of boricacid in water (30 mL) were taken together in a pressure reactor andheated at 90° C. for 3 days. Progress of the reaction was monitored byTLC and Mass spectra. The reaction mixture extracted intodichloromethane (DCM), washed successively with NaHCO₃ solution andbrine, dried over anhydrous sodium sulfate. Solvent was removed in vacuoand residue thus obtained was purified by silica gel (2 Kg) columnchromatography (gradient elution-0:5:95-10:10:80% TEA/MeOH/DCM) toobtain 117 as a pale yellow solid (83.8 g, 42%). MS: C₇₆H₁₅₀N₈O₈ Cal.1303.16. Found 1304.20 (M⁺). The material was compared with authenticsample TLC (qualitative), HPLC and Mass spectra. MS: C₈₁H₁₆₃N₉O₅ Cal.1342.28. Found 1343.30 (M⁺).

Step 7. Synthesis of the Hydrochloride Salt 7

The amine 117 (54 g, 40 mmol) was dissolved ethanol (100 mL) and to it200 mL of 2M HCl in ether was added and the mixture was stirred at roomtemperature overnight. Nitrogen was bubbled to the reaction mixture andthe outlet was passed through dryrite and to a 10% solution of KOH.After 30 minute, the reaction mixture was concentrated to dryness andthe residue was re-dissolved in 500 mL of anhydrous ethanol and themixture was concentrated in a rotary evaporator. This process was againrepeated once again and the thus obtained residue was dried in a vacuumoven at 43° C. overnight. The pure product was isolated as a creampowder (59.5 g, 99%).

Example 40 Regioselective Synthesis of Cationic Lipid 7—Strategy 2

Method 1

Step 1: Triethylenetetramine, 1 (200 g g, 1.37 mol, purchased fromSigma-Aldrich) in acetonitrile (2 L) in a 4 neck 5 L flask with overheadstirrer was cooled over an ice bath under constant stirring. Ethyltrifluoroacetate (388.5 g, 2.74 mol) was added to the stirring solutionand stirred for 20 h. Solvent and volatiles were removed under reducedpressure; the residue was triturated with a mixture of DCM/Hexane andfiltered to get 101 as white solid (429 g, 93%). The product thusobtained could be used for the next reaction without furtherpurification. MS: C₁₀H₁₆F₆N₄O₂ Cal. 338.12. Found 339.0 (M⁺).

Step 2: Crude compound 101 (427 g, 1.26 mol) was dissolved in a mixtureof solvents (3 L, THF/DCM (1:2)) and stirred over an ice-water bath.Di-tert-butyl dicarbonate ((Boc)₂O, 270 g, 1.26 mol, purchased fromSigma Aldrich) and DIEA (500 mL, 2.86 mol) were added to the reactionmixture and stirred overnight. Solvents were removed and the residue wasextracted into dichloromethane (DCM, 1000 mL), washed successively withNaHCO3 solution (500 mL), water (500 mL×2) and brine, dried overanhydrous sodium sulfate. Solvents were removed in vacuo and residuethus obtained was triturated with DCM/Hexane (2:1) and filtered.Solvents were removed and the residue was dried under high vacuum to getthe compound 102 as gummy liquid (523 g).

Part of the compound 102 was purified by silica gel chromatography(gradient elution, Ethyl acetate, followed by 3-10% MeOH/DCM) to obtaincompound 102 as gummy liquid (102.00 g). ¹H NMR for 102 (DMSO-d6, 400MHz) δ=9.60-9.10(m, 3H), 3.35-3.25(m, 4H), 3.25-3.20(2, 2H),3.20-3.10(m, 2H), 2.68-2.58(m, 4H), 1.35(s, 9H). MS: C₁₅H₂₄F₆N₄O₄ Cal.438.17. Found 439.20(M⁺).

Step 3: Purified compound 102 (102.0 g, 233.40 mmol) was dissolved inEthanol/Methyl amine (400 ml, 33 wt % methylamine solution in EtOH) atambient temperature in a pressure reactor. The mixture was warmed to 90°C. and stirred for two days. Reaction was monitored by mass spectra. Allthe solvents were removed under reduced pressure and the residue wassubjected to high vacuum at 80° C. to yield the product 103 (58.00 g,99%) as gummy liquid and this compound could be used for the nextreaction with out further purification. ¹H NMR (CDCl₃, 400 MHz)δ=3.20-3.00(m, 4H), 2.62-2.38 (m, 8H), 1.32(s, 9H). MS: C₁₁H₂₆N₄O₂ Cal.246.21. Found 247.20(M⁺).

Step 4: Triamine 103 (56.00 g, 227.64 mmol), N-dodecylacrylamide (327.00g, 1365 mmol) and saturated solution of boric acid in water (50 mL) weretaken together in a pressure reactor and heated at 90° C. for 6 days.The reaction was monitored by TLC and Mass spectra. The reaction mixtureextracted into dichloromethane (DCM), washed successively with NaHCO₃solution (400 mL) and dried over anhydrous sodium sulfate. Solvent wasremoved in vacuo and residue thus obtained was purified by silica gelcolumn chromatography (gradient elution-Ethyl acetate then 3-10%MeOH/DCM) to obtain 104 as a pale yellow solid (186 g, 57%). ¹H NMR(CDCl₃, 400 MHz) δ=7.20(bs, 1H), 7.05(bs, 1H), 6.85(bs, 1H), 6.74(bs,1H), 3.25-3.03 (m, 12H), 2.80-2.60(m, 8H), 2.55-2.21(m, 12H)1.52-1.45(m, 10H), 1.42 (s, 9H), 1.34-1.20(m, 100H), 0.87(t, J=6.5 Hz,15H). MS: C₈₆H₁₇₁N₉O₇ Cal. 1442.33. Found 1443.30(M⁺).

Step 5: 4M HCl in dioxane (400 mL) was added into a solution of compound105 (184.00 g, 127.23 mmol) in dioxane (300 mL). The reaction mixturewas then allowed to stir for overnight. The reaction was monitored byMass spectra. Excess HCl was removed by passing nitrogen through thesolution. Solvents were removed under vacuum and residue was coevaporated three times with ethanol (500 mL×3) to yield a pale yellowgummy solid 7 (186.00 g, 98%) as tetrahydrochloride salt. The materialwas compared with authentic sample TLC (qualitative), HPLC and Massspectra. MS: C₈₁H₁₆₃N₉O₅ Cal. 1342.28. Found 1343.30 (M⁺).

Method 2

Compound 102 was prepared as described in Method 1: steps 1 and 2. Thecrude product obtained from step 2 of Method 1 was used for the nextreaction without further purification.

Step 1: Compound 102 (103.45 g, 238.90 mmol, crude compound from step 2,Method 1 was dissolved in Ethanol/Methyl amine (400 ml, 33 wt %methylamine solution in EtOH) at ambient temperature in a pressurereactor. The mixture was warmed to 90° C. and stirred for two days.Reaction was monitored by mass spectra. All the solvents were removedunder reduced pressure and the residue was subjected to high vacuum at80° C. over a water bath to yield the product 103 (63.50 g) as paleyellow gummy liquid and this compound could be used for the nextreaction with out further purification.

Step 4: Triamine 103 (63.50 g, 238 mmol), N-dodecylacrylamide (320.00 g,1338 mmol) and saturated solution of boric acid in water (50 mL) weretaken together in a pressure reactor and heated at 90° C. for 6 days asdescribed in step 4, Method 1. The reaction was monitored by TLC andMass spectra. The reaction mixture extracted into dichloromethane (DCM),washed successively with NaHCO₃ solution (400 mL) and dried overanhydrous sodium sulfate. Solvent was removed in vacuo and residue thusobtained was purified by silica gel column chromatography (gradientelution-Ethyl acetate then 3-10% MeOH/DCM) to obtain 104 as a paleyellow solid (65.2 g, 20%).

Step 5: 2M HCl in ether (800 mL) was added to compound 105 (65.00 g, 45mmol). The reaction mixture was then allowed to stir for overnight. Thereaction was monitored by Mass spectra. Excess HCl was removed bypassing nitrogen through the solution. Solvents were removed undervacuum and residue was co evaporated three times with ethanol (500 mL×3)to yield a pale yellow gummy solid 7 (66 g, 98%) as tetra hydrochloridesalt. The material was compared with authentic sample TLC (qualitative),HPLC and Mass spectra. MS: C₈₁H₁₆₃N₉O₅ Cal. 1342.28. Found 1343.30 (M⁺).

Method 3

Compound 102 was prepared as described in Method 1: steps 1 and 2. Thecrude product obtained from step 2 of Method 1 was used for the nextreaction without further purification.

Step 3: Compound 102 (105.20 g, 240 mmol, crude compound from method I)was dissolved in Ethanol/Methyl amine (400 ml, 33 wt % methylaminesolution in EtOH) at ambient temperature in a pressure reactor. Themixture was warmed to 90° C. and stirred for two days. Reaction wasmonitored by mass spectra. All the solvents were removed under reducedpressure and the residue was subjected to high vacuum at 80° C. over awater bath to yield the product 103 (64.70 g) as pale yellow gummyliquid and this compound could be used for the next reaction with outfurther purification.

Step 4: Triamine 103 (64.70 g, 240 mmol), N-dodecylacrylamide (370.00 g,1569 mmol) and saturated solution of boric acid in water (50 mL) weretaken together in a pressure reactor and heated at 90° C. for 6 days.The reaction was monitored by TLC and Mass spectra. The reaction mixtureextracted into dichloromethane (DCM), washed successively with NaHCO₃solution (400 mL) and dried over anhydrous sodium sulfate. Solvent wasremoved in vacuo and residue thus obtained was purified by silica gelcolumn chromatography (gradient elution-Ethyl acetate then 3-10%MeOH/DCM) to obtain 104 as a pale yellow solid (192 g).

Step 5: The desired compound 7 was obtained as hydrochloride salt fromcompound 104 as described in step 5, Method 1 of Example 40. Compound 7:194 g (98%) as tetrahydrochloride salt. The material was compared withauthentic sample TLC (qualitative), HPLC and Mass spectra. MS:C₈₁H₁₆₃N₉O₅ Cal. 1342.28. Found 1343.30 (M⁺).

Example 41 Comparison of Activity of siRNA Formulated into VariousAssociation Complexes Having Differing Peg-Lipid Moieties

The effectiveness of lipid compositions can be tested by determining therelative ability of a lipid to deliver an siRNA moiety to a target. Forexample, the silencing of a target indicates that the siRNA is deliveredinto the cell. Applicants have compared association complexes thatinclude one of 13 different PEG-lipid moieties as provided in FIG. 5,together with siRNA that is used to silence Factor VII (FVII).

PEG-lipids 1-13 were synthesized using the following procedures:

Preparation of compound 5: 1,2-Di-O-tetradecyl-sn-glyceride 1 (30 g,61.80 mmol) and N,N′-succinimidylcarboante (DSC, 23.76 g, 1.5 eq) weretaken in dichloromethane (DCM, 500 mL) and stirred over an ice watermixture. Triethylamine (25.30 mL, 3 eq) was added to stirring solutionand subsequently the reaction mixture was allowed to stir overnight atambient temperature. Progress of the reaction was monitored by TLC. Thereaction mixture was diluted with DCM (400 mL) and the organic layer waswashed with water (2×500 mL), aqueous NaHCO₃ solution (500 mL) followedby standard work-up. Residue obtained was dried at ambient temperatureunder high vacuum overnight. After drying the crude carbonate 3 thusobtained was dissolved in dichloromethane (500 mL) and stirred over anice bath. To the stirring solution mPEG₂₀₀₀-NH₂ (4, 103.00 g, 47.20mmol, purchased from NOF Corporation, Japan) and anhydrous pyridine (80mL, excess) were added under argon. The reaction mixture was thenallowed stir at ambient temperature overnight. Solvents and volatileswere removed under vacuum and the residue was dissolved in DCM (200 mL)and charged on a column of silica gel packed in ethyl acetate. Thecolumn was initially eluted with ethyl acetate and subsequently withgradient of 5-10% methanol in dichloromethane to afford the desiredPEG-Lipid 5 as a white solid (105.30 g, 83%). ¹H NMR (CDCl₃, 400 MHz)δ=5.20-5.12(m, 1H), 4.18-4.01(m, 2H), 3.80-3.70(m, 2H), 3.70-3.20(m,—O—CH₂—CH₂—O—, PEG-CH₂), 2.10-2.01(m, 2H), 1.70-1.60 (m, 2H),1.56-1.45(m, 4H), 1.31-1.15(m, 48H), 0.84(t, J=6.5 Hz, 6H). MS rangefound: 2660-2836.

Preparation of 4b: 1,2-Di-O-hexadecyl-sn-glyceride 1b (1.00 g, 1.848mmol) and DSC (0.710 g, 1.5 eq) were taken together in dichloromethane(20 mL) and cooled down to 0° C. in an ice water mixture. Triethylamine(1.00 mL, 3 eq) was added to that and stirred overnight. The reactionwas followed by TLC, diluted with DCM, washed with water (2 times),NaHCO₃ solution and dried over sodium sulfate. Solvents were removedunder reduced pressure and the residue 2b under high vacuum overnight.This compound was directly used for the next reaction without furtherpurification. MPEG₂₀₀₀-NH₂ 3 (1.50 g, 0.687 mmol, purchased from NOFCorporation, Japan) and compound from previous step 2b (0.702 g, 1.5 eq)were dissolved in dichloromethane (20 mL) under argon. The reaction wascooled to 0° C. Pyridine (1 mL, excess) was added to that and stirredovernight. The reaction was monitored by TLC. Solvents and volatileswere removed under vacuum and the residue was purified by chromatography(first Ethyl acetate then 5-10% MeOH/DCM as a gradient elution) to getthe required compound 4b as white solid (1.46 g, 76%). ¹H NMR (CDCl₃,400 MHz) δ=5.17(t, J=5.5 Hz, 1H), 4.13(dd, J=4.00 Hz, 11.00 Hz, 1H),4.05(dd, J=5.00 Hz, 11.00 Hz, 1H), 3.82-3.75(m, 2H), 3.70-3.20(m,—O—CH₂—CH₂—O—, PEG-CH₂), 2.05-1.90(m, 2H), 1.80-1.70 (m, 2H),1.61-1.45(m, 6H), 1.35-1.17(m, 56H), 0.85(t, J=6.5 Hz, 6H). MS rangefound: 2716-2892.

Preparation of 4c: 1,2-Di-O-octadecyl-sn-glyceride 1c (4.00 g, 6.70mmol) and DSC (2.58 g, 1.5 eq) were taken together in dichloromethane(60 mL) and cooled down to 0° C. in an ice water mixture. Triethylamine(2.75 mL, 3 eq) was added to that and stirred overnight. The reactionwas followed by TLC, diluted with DCM, washed with water (2 times),NaHCO₃ solution and dried over sodium sulfate. Solvents were removedunder reduced pressure and the residue under high vacuum overnight. Thiscompound was directly used for the next reaction with furtherpurification. MPEG₂₀₀₀-NH₂ 3 (1.50 g, 0.687 mmol, purchased from NOFCorporation, Japan) and compound from previous step 2c (0.760 g, 1.5 eq)were dissolved in dichloromethane (20 mL) under argon. The reaction wascooled to 0° C. Pyridine (1 mL, excess) was added to that and stirredovernight. The reaction was monitored by TLC. Solvents and volatileswere removed under vacuum and the residue was purified by chromatography(first Ethyl acetate then 5-10% MeOH/DCM as a gradient elution) to getthe required compound 4 c as white solid (0.92 g, 48%). ¹H NMR (CDCl₃,400 MHz) δ=5.22-5.15(m, 1H), 4.16 (dd, J=4.00 Hz, 11.00 Hz, 1H),4.06(dd, J=5.00 Hz, 11.00 Hz, 1H), 3.81-3.75(m, 2H), 3.70-3.20(m,—O—CH₂—CH₂—O—, PEG-CH₂), 1.80-1.70 (m, 2H), 1.60-1.48(m, 4H),1.31-1.15(m, 64H), 0.85(t, J=6.5 Hz, 6H). MS range found: 2774-2948.

Preparation of compound 6a: 1,2-Di-O-tetradecyl-sn-glyceride 1a (1.00 g,2.06 mmol), succinic anhydride (0.416 g, 2 eq) and DMAP (0.628 g, 2.5eq) were taken together in dichloromethane (20 mL) and stirredovernight. The reaction was followed by TLC, diluted with DCM, washedwith cold dilute citric acid, water and dried over sodium sulfate.Solvents were removed under reduced pressure and the residue under highvacuum overnight. This compound was directly used for the next reactionwith further purification. MPEG₂₀₀₀-NH₂ 3 (1.50 g, 0.687 mmol, purchasedfrom NOF Corporation, Japan), compound from previous step 5a (0.66 g,1.12 eq) and HBTU (0.430 g, 1.13 mmol) were dissolved in a mixture ofdichloromethane/DMF (2:1, 20 mL) under argon. DIEA (0.358 mL, 3 eq.) wasadded to that and stirred overnight. The reaction mixture wastransferred to a large flask and removed the solvents and volatilesunder reduced pressure. The residue was dried under high vacuumovernight and purified by chromatography (first ethyl acetate then 5-10%MeOH/DCM as a gradient elution) to get the required compound 6a as whitesolid (0.822 g, 43%). ¹H NMR (CDCl₃, 400 MHz) δ=6.34-6.30(m, 1H),4.16(dd, J=4.00 Hz, 11.00 Hz, 1H), 4.08(dd, J=5.00 Hz, 11.00 Hz, 1H),3.82-3.78(m, 2H), 3.70-3.30(m, —O—CH₂—CH₂—O—, PEG-CH₂), 2.64 (t, J=7.00Hz, 2H), 2.43(t, J=6.80 Hz, 2H), 1.76-1.72 (m, 2H), 1.56-1.48(m, 4H),1.34-1.16(m, 48H), 0.85(t, J=6.5 Hz, 6H). MS range found 2644-2804.

Preparation of compound 6b: 1,2-Di-O-hexadecyl-sn-glyceride 1b (1.00 g,1.848 mmol), succinic anhydride (0.0.369 g, 2 eq) and DMAP (0.563 g, 2.5eq) were taken together in dichloromethane (20 mL) and stirredovernight. The reaction was followed by TLC, diluted with DCM, washedwith cold dilute citric acid, water and dried over sodium sulfate.Solvents were removed under reduced pressure and the residue under highvacuum overnight. This compound was directly used for the next reactionwith further purification. MPEG₂₀₀₀-NH₂ 3 (1.50 g, 0.687 mmol, purchasedfrom NOF Corporation, Japan), compound from previous step 5b (0.66 g,1.03 mmol) and HBTU (0.400 g, 1.05 mmol) were dissolved in a mixture ofdichloromethane/DMF (2:1, 20 mL) under argon. DIEA (0.358 mL, 3 eq.) wasadded to that and stirred overnight. The reaction mixture wastransferred to a large flask and removed the solvents and volatilesunder reduced pressure. The residue was dried under high vacuumovernight and purified by chromatography (first ethyl acetate then 5-10%MeOH/DCM as a gradient elution) to get the required compound 6b as whitesolid (0.300 g, 16%). ¹H NMR (CDCl₃, 400 MHz) δ=6.33-6.28(m, 1H),4.18(dd, J=4.00 Hz, 11.00 Hz, 1H), 4.08(dd, J=5.00 Hz, 11.00 Hz, 1H),3.82-3.76(m, 2H), 3.70-3.30(m, —O—CH₂—CH₂—O—, PEG-CH₂), 2.65 (t, J=7.08Hz, 2H), 2.44(t, J=6.83 Hz, 2H), 1.76-1.68 (m, 2H), 1.57-1.48(m, 4H),1.32-1.17(m, 56H), 0.86(t, J=6.6 Hz, 6H). MS range found: 2640-2822.

Preparation of compound 6c: 1,2-Di-O-octadecyl-sn-glyceride 1c (5.00 g,8.37 mmol), succinic anhydride (1.70 g, 2 eq) and DMAP (2.55 g, 2.5 eq)were taken together in dichloromethane (50 mL) and stirred overnight.The reaction was followed by TLC, diluted with DCM, washed with colddilute citric acid, water and dried over sodium sulfate. Solvents wereremoved under reduced pressure and the residue under high vacuumovernight. This compound was directly used for the next reaction withfurther purification. MPEG₂₀₀₀-NH₂ 3 (1.50 g, 0.687 mmol, purchased fromNOF Corporation, Japan), compound from previous step 5c (0.718 g, 1.03mmol) and HBTU (0.410 g, 1.08 mmol) were dissolved in a mixture ofdichloromethane/DMF (2:1, 20 mL) under argon. DIEA (0.350 mL, 3 eq.) wasadded to that and stirred overnight. The reaction mixture wastransferred to a large flask and removed the solvents and volatilesunder reduced pressure. The residue was dried under high vacuumovernight and purified by chromatography (first ethyl acetate then 5-10%MeOH/DCM as a gradient elution) to get the required compound 6c as whitesolid (1.1 g, 56%). ¹H NMR (CDCl₃, 400 MHz) δ=6.38-6.33(m, 1H), 4.19(dd,J=4.00 Hz, 11.00 Hz, 1H), 4.07(dd, J=5.00 Hz, 11.00 Hz, 1H),3.81-3.74(m, 2H), 3.70-3.20(m, —O—CH₂—CH₂—O—, PEG-CH₂), 2.63 (t, J=7.03Hz, 2H), 2.43(t, J=6.87 Hz, 2H), 1.76-1.68 (m, 2H), 1.57-1.48(m, 4H),1.32-1.17(m, 64H), 0.86(t, J=6.60 Hz, 6H). MS range found: 2680-2922

Preparation of compound 8a: 1,2-Di-O-tetradecyl-sn-glyceride 1a (0.300g, 0.618 mmol), MPEG-Succinate 7 (1.00 g, 0.476 mmol, purchased from NOFCorporation, Japan), DCC (0.127 g, 1.3 eq) and DMAP (0.058 g, 0.476mmol) were taken in dichloromethane (20 mL) under argon and stirredovernight. Reaction was monitored by TLC. The reaction mixture wascooled to 0° C. after stirring overnight and filtered off theprecipitated solid. Volatiles and solvents were removed under reducedpressure and the resulting residue was purified by chromatography (firsteluted with EtOAc, followed by 5-10% DCM/MeOH gradient elution) to getthe compound 8a as a white solid (0.590 g, 48%). ¹H NMR (CDCl₃, 400 MHz)δ=4.25-4.18(m, 2H), 4.08(dd, J=5.60 Hz, 11.50 Hz, 1H), 3.80-3.73(m, 2H),3.70-3.30(m, —O—CH₂—CH₂—O—, PEG-CH₂), 1.56-1.47(m, 4H), 1.30-1.15(m,48H), 0.85(t, J=6.60 Hz, 6H). MS range found: 2440-2708

Preparation of compound 8b: 1,2-Di-O-hexadecyl-sn-glyceride 1b 0.334 g,0.618 mmol), MPEG-Succinate 7 (1.00 g, 0.476 mmol, purchased from NOFCorporation, Japan), DCC (0.127 g, 1.3 eq) and DMAP (0.058 g, 0.476mmol) were taken in dichloromethane (20 mL) under argon and stirredovernight. Reaction was monitored by TLC. The reaction mixture wascooled to 0° C. after stirring overnight and filtered off theprecipitated solid. Volatiles and solvents were removed under reducedpressure and the resulting residue was purified by chromatography (firsteluted with EtOAc, followed by 5-10% DCM/MeOH gradient elution) to getthe compound 8b as a white solid (0.930 g, 74%). ¹H NMR (CDCl₃, 400 MHz)δ=4.25-4.17(m, 2H), 4.09(dd, J=5.50 Hz, 11.50 Hz, 1H), 3.81-3.73(m, 2H),3.70-3.30(m, —O—CH₂—CH₂—O—, PEG-CH₂), 1.58-1.47(m, 4H), 1.30-1.17(m,56H), 0.86(t, J=6.60 Hz, 6H). MS range found: 2452-2760.

Preparation of compound 8c: 1,2-Di-O-octadecyl-sn-glyceride 1c (0.369 g,0.618 mmol), MPEG-Succinate 7 (1.00 g, 0.476 mmol, purchased from NOFCorporation, Japan), DCC (0.127 g, 1.3 eq) and DMAP (0.058 g, 0.476mmol) were taken in dichloromethane (20 mL) under argon and stirredovernight. Reaction was monitored by TLC. The reaction mixture wascooled to 0° C. after stirring overnight and filtered off theprecipitated solid. Volatiles and solvents were removed under reducedpressure and the resulting residue was purified by chromatography (firsteluted with EtOAc, followed by 5-10% DCM/MeOH gradient elution) to getthe compound 8c as a white solid (0.960 g, 75%). ¹H NMR (CDCl₃, 400 MHz)δ=4.27-4.20(m, 2H), 4.10(dd, J=5.80 Hz, 11.50 Hz, 1H), 3.83-3.74(m, 2H),3.70-3.35(m, —O—CH₂—CH₂—O—, PEG-CH₂), 1.54-1.46(m, 4H), 1.30-1.17(m,64H), 0.86(t, J=6.60 Hz, 6H). MS range found: 2508-2816.

Preparation of compound 10a: 1,2-Dimyristoyl-sn-glycerol 9a (0.317 g,0.618 mmol), MPEG-Succinate 7 (1.00 g, 0.476 mmol, purchased from NOFCorporation, Japan), DCC (0.127 g, 1.3 eq) and DMAP (0.058 g, 0.476mmol) were taken in dichloromethane (20 mL) under argon and stirredovernight. Reaction was monitored by TLC. The reaction mixture wascooled to 0° C. after stirring overnight and filtered off theprecipitated solid. Volatiles and solvents were removed under reducedpressure and the resulting residue was purified by chromatography (firsteluted with EtOAc, followed by 5-10% DCM/MeOH gradient elution) to getthe compound 10a as a white solid (0.960 g, 78%). ¹H NMR (CDCl₃, 400MHz) δ=5.26-5.20(m, 1H), 4.30-4.08(m, 6H), 3.81-3.73(m, 2H),3.70-3.40(m, —O—CH₂—CH₂—O—, PEG-CH₂), 2.65-2.60(m, 4H), 2.35-2.28 (m,4H), 1.63-1.52(m, 4H), 1.30-1.15(m, 44H), 0.86(t, J=6.60 Hz, 6H). MSrange found: 2468-2732.

Preparation of compound 10b: 1,2-Dipalmitoyl-sn-glycerol 9b (0.352 g,0.618 mmol), MPEG-Succinate 7 (1.00 g, 0.476 mmol, purchased from NOFCorporation, Japan), DCC (0.127 g, 1.3 eq) and DMAP (0.058 g, 0.476mmol) were taken in dichloromethane (20 mL) under argon and stirredovernight. Reaction was monitored by TLC. The reaction mixture wascooled to 0° C. after stirring overnight and filtered off theprecipitated solid. Volatiles and solvents were removed under reducedpressure and the resulting residue was purified by chromatography (firsteluted with EtOAc, followed by 5-10% DCM/MeOH gradient elution) to getthe compound 10b as a white solid (1.02 g, 81%). ¹H NMR (CDCl₃, 400 MHz)δ=5.26-5.19(m, 1H), 4.30-4.05(m, 6H), 3.80-3.40(m, —O—CH₂—CH₂—O—,PEG-CH₂), 2.65-2.60(m, 4H), 2.33-2.24(m, 4H), 1.63-1.50(m, 4H),1.30-1.15(m, 52H), 0.85(t, J=6.60 Hz, 6H). MS range found: 2524-2792.

Preparation of compound 10c: 1,2-Distearoyl-sn-glycerol 9c (0.387 g,0.618 mmol), MPEG-Succinate 7 (1.00 g, 0.476 mmol, purchased from NOFCorporation, Japan), DCC (0.127 g, 1.3 eq) and DMAP (0.058 g, 0.476mmol) were taken in dichloromethane (20 mL) under argon and stirredovernight. Reaction was monitored by TLC. The reaction mixture wascooled to 0° C. after stirring overnight and filtered off theprecipitated solid. Volatiles and solvents were removed under reducedpressure and the resulting residue was purified by chromatography (firsteluted with EtOAc, followed by 5-10% DCM/MeOH gradient elution) to getthe compound 10c as a white solid (1.04 g, 80%). ¹H NMR (CDCl₃, 400 MHz)δ=5.26-5.19(m, 1H), 4.30-4.05(m, 6H), 3.80-3.40(m, —O—CH₂—CH₂—O—,PEG-CH₂), 2.66-2.59(m, 4H), 2.31-2.26(m, 4H), 1.63-1.52(m, 4H),1.30-1.15(m, 52H), 0.85(t, J=6.60 Hz, 6H). MS range found: 2540-2844.

Preparation of compound 13: mPEG₂₀₀₀-OH 11 (6.00 g, 3 mmol, purchasedfrom Sigma-Aldrich), Cholesterol hemisuccinate 12 (1.50 g, 3.08 mmolmmol) and HBTU (1.23 g, 3.23 mmol) were dissolved in a mixture ofdichloromethane/DMF (2:1, 100 mL) under argon. DIEA (1.60 mL, 3 eq.) wasadded to that and stirred overnight. Solvents and volatiles were removedunder reduced pressure. The residue was dried under high vacuumovernight and purified by chromatography (first ethyl acetate then 5-10%MeOH/DCM as a gradient elution) to get the required compound 13 as whitesolid (5.05 g, 68%). ¹H NMR (CDCl₃, 400 MHζ) δ=5.35-5.25(m, 1H),4.60-4.50(m, 1H), 4.22-4.18(m, 2H), 3.80-3.76(m, 2H), 3.72-3.40(m,—O—CH₂—CH₂—O—, PEG-CH₂), 2.64-2.56(m, 4H), 2.31-2.20(m, 3H), 2.01-0.8(m,44H).MS range found: 2390-2654.

Example 42 Targeted PEG-Lipids

Preparation of 19:

Step 1: Compound 14 (2.00 g, 1.01 mmol) and cholesterol chloroformate 15(0.453 g, 1.01 mmol) were taken together in dichloromethane (20 mL). Themixture was cooled in an ice-water bath. Triethylamine (0.448 ml) wasadded and the reaction mixture was stirred overnight. Reaction wasmonitored by TLC. Solvent was removed and the residue was purified bysilica gel chromatography (Ethyl acetate followed by 5-10% MeOH/DCM) toget the desired compound 16 (1.10 g, 45.40%). ¹H NMR (CDCl₃, 400 MHz)δ=5.35(m, 1H), 5.15(m, 1H), 3.40-3.85(m, O—CH₂—CH₂—O), 3.10-3.25(m,10H), 0.80-2.38(m, 44H, Cholesterol). MS range found: 2220-2490.

Step 2: Compound 16 (1.00 g, 0.417 mmol), 17 (0.235 g, 0.542 mmol) andHBTU (0.190 g, 0.5 mmol) were taken in a mixture of DCM/DMF (20 mL,2:1). To that DIEA was added and stirred overnight. Reaction wasmonitored by TLC, solvents were removed under reduced pressure and theresidue was purified by chromatography (5-10% MeOH/DCM) to get thedesired compound 18 (1.02 g, 87%). ¹H NMR (DMSO-d6, 400 MHz) δ=7.52(d,J=8.06 Hz, 1H), 7.33(t, J=7.02 Hz, 1H), 7.25(t, J=7.32 Hz, 1H), 5.27(m,1H), 5.18(d, J=3.2 Hz, 1H), 4.92(dd, J=3.17, 11.23 Hz, 1H), 4.43(m, 1H),3.60-4.02(m, 5H), 3.20-3.55(m, O—CH₂—CH₂—O), 2.90-3.10(m, 10H), 2.05(s,3H), 1.96(s, 3H), 1.84(s, 3H), 1.77(s, 3H), 0.80-2.38(m, 44H,Cholesterol). MS range found: 2680-2990.

Step 3: Compound 18 (1.02 g, 0.362 mmol) was dissolved in a mixture ofMeOH/DCM (10 mL) to that 0.5 M solution of NaOMe in methanol (excess)was added and stirred overnight. Progress of the reaction was monitoredby TLC. The mixture was neutralized with AcOH. Solvents were removedunder vacuum and the residue was purified by chromatography (5-10%MeOH/DCM) to get compound 19 (280 mg, 30%). ¹H NMR (CDCl₃, 400 MHz)δ=5.38(m, 1H), 4.02-4.06(m, 7H), 3.30-3.80(m, O—CH₂—CH₂—O), 3.20-3.29(m,8H), 2.08(s, 3H), 0.80-2.38(m, 44H, Cholesterol). MS range found:2600-2900.

Example 43 Targeted PEG-Lipids

Preparation of 23:

Step 1: Compound 14 (2.00 g, 1.01 mmol) and compound 20 (0.453 g, 1.01mmol) were taken together in dichloromethane (20 mL). The mixture wascooled in an ice-water bath. Pyridine (1 mL, excess) was added and thereaction mixture was stirred overnight. Reaction was monitored by TLC.Solvent was removed and the residue was purified by silica gelchromatography (Ethyl acetate followed by 5-10% MeOH/DCM) to get thedesired compound 21 (400 mg, 15%). ¹H NMR (CDCl₃, 400 MHz) δ=5.20(m,1H), 4.05-4.20(m, 2H), 3.20-3.80(m, O—CH₂—CH₂—O), 1.70-1.82(m, 4H),1.50-1.61(m, 2H), 1.18-1.38(m, 60H), 0.87(t, J=6.30 Hz, 6H). MS rangefound: 2400-2750.

Step 2: Compound 21 (0.415 g, 0.159 mmol), 17 (0.100 g, 1.3 eq) and HBTU(0.90 g, 1.15 eq) were taken in a mixture of DCM/DMF (20 mL, 2:1). Tothat DIEA (0.2 mL) was added and stirred overnight. Reaction wasmonitored by TLC, solvents were removed under reduced pressure and theresidue was purified by chromatography (3-10% MeOH/DCM) to get thedesired compound 22 (0.450 g, 94%). ¹H NMR (CDCl₃, 400 MHz) δ=6.21(d,J=8.70 Hz, 1H), 5.33(d, J=2.70 Hz, 1H), 5.15-5.20(m, 2H), 4.55(d, J=8.15Hz, 1H), 4.01-4.20(m, 4H), 3.20-3.90(m, O—CH₂—CH₂—O), 2.14(s, 3H),2.03(s, 3H), 1.99(s, 3H), 1.93(s, 3H), 1.70-1.82(m, 4H), 1.50-1.61(m,4H), 1.17-1.38(m, 60H), 0.86(t, J=6.32 Hz, 6H). MS range found:2800-3200.

Step 3: Compound 22 (0.450 g, 0.359 mmol) was dissolved in a mixture ofMeOH/DCM (5 mL) to that 0.5 M solution of NaOMe in methanol (excess) wasadded and stirred overnight. Progress of the reaction was monitored byTLC. The mixture was neutralized with AcOH. Solvents were removed undervacuum and the residue was purified by chromatography (5-10% MeOH/DCM)to get compound 23 (365 mg, 85%). ¹H NMR (CDCl₃, 400 MHz) δ=5.18(m, 1H),4.05-4.20(m, 4H), 3.20-3.90(m, O—CH₂—CH₂—O), 2.05(s, 3H), 1.71-1.80(m,4H), 1.50-1.61(m, 4H), 1.17-1.38(m, 60H), 0.87(t, J=6.32 Hz, 6H). MSrange found: 2760-3000.

As provided in FIG. 6, the formulations, when administered to a subject,provided a varying degree of silencing of FVII. For example, formulation3 provided a relative high degree of silencing of FVII, as didformulation 5, 6, and 12.

Example 44 Tolerability of Formulation LNP01 as Dosed in Mice

Empty liposomes with composition ND98:cholesterol:PEG-C14=42:48:10(molar ratio) were prepared as described in Example 45. Differentamounts of siRNA were then added to the pre-formed, extruded emptyliposomes to yield formulations with initial total excipient:siRNAratios of 30:1, 20:1, 15:1, 10:1, and 5:1 (wt:wt). Preparation of aformulation at a total excipient:siRNA ratio of 5:1 results in an excessof siRNA in the formulation, saturating the lipid loading capacity.Excess siRNA was then removed by tangential flow filtration using a100,000 MWCO membrane against 5 volumes of PBS. The resultingformulations were then administered to C57BL/6 mice via tail veininjection at 10 mg/kg siRNA dose. Tolerability of the formulations wasassessed by measuring the body weight gain of the animals 24 h and 48 hpost administration of the formulation, the results of which areprovided in FIG. 7.

Example 45 Formation of Association Complexes by First Forming UnloadedComplexes and then Treating the Unloaded Complexes with siRNA andAdministration of Association Complexes Including Two Therapeutic Agents

Association complexes having two different nucleic acid moieties wereprepared as follows. Stock solutions of ND98, cholesterol, and PEG-C14in ethanol were prepared at the following concentrations: 133 mg/mL, 25mg/mL, and 100 mg/mL for ND98, cholesterol, and PEG-C14, respectively.The lipid stocks were then mixed to yield ND98:cholesterol:PEG-C14 molarratios of 42:48:10. This mixture was then added to aqueous bufferresulting in the spontaneous formulation of lipid nanoparticles in 35%ethanol, 100 mM sodium acetate, pH 5. The unloaded lipid nanoparticleswere then passed twice through a 0.08 μm membrane (Whatman, Nucleopore)using an extruder (Lipex, Northern Lipids) to yield unimodal vesicles20-100 nm in size. The appropriate amount of siRNA in 35% ethanol wasthen added to the pre-sized, unloaded vesicles at a totalexcipient:siRNA ratio of 7.5:1 (wt:wt). The resulting mixture was thenincubated at 37° C. for 30 min to allow for loading of siRNA into thelipid nanoparticles. After incubation, ethanol removal and bufferexchange was performed by either dialysis or tangential flow filtrationagainst PBS. The final formulation was then sterile filtered through a0.2 μm filter. A flow chart demonstrating the order of addition ofexhipients and therapeutic agents is provided in FIG. 8.

A 1:1 mixture of siRNAs targeting ApoB and Factor VII were formulated asdescribed in Example 44. Separately, the same ApoB- and FactorVII-targeting siRNAs were individually formulated as described inExample 31. The three formulations were then administered at varyingdoses in an injection volume of 10 μL/g animal body weight. Forty-eighthours after administration, serum samples were collected by retroorbitalbleed, animals were sacrificed, and livers were harvested. Serum FactorVII concentrations were determined using a chromogenic diagnostic kit(Coaset Factor VII Assay Kit, DiaPharma) according to manufacturerprotocols. Liver mRNA levels of ApoB and Factor VII were determinedusing a branched-DNA (bDNA) assay (Quantigene, Panomics), the results ofwhich are provided in FIG. 9. No evidence of inhibition between the twotherapeutic agents was observed. Rather, both of the therapeutic agentsdemonstrated effectiveness when administered.

Example 46 Methods of Making Association Complexes Using PreformedVesicles

Lipid Stock Preparation

Stock solutions of lipidoid ND98.4HCl (MW 1487), cholesterol, andPEG-C14 were prepared in ethanol at the following concentrations: 133mg/mL, 25 mg/mL, and 100 mg/mL for ND98, cholesterol, and PEG-C14,respectively. Stock solutions were warmed at 50° C. to assist in bringlipids into solution.

Empty Vesicle Preparation

The lipid stocks were then mixed according to the volumes listed belowto yield ND98:cholesterol:PEG-C14 molar ratios of 42:48:10. An aqueousmixture was also prepared according to the volumes listed in the tablebelow.

Volume Lipid Mixture (mL) ND98 Cholesterol PEG Total  56.250 90.00031.500 177.750 Aqueous Mixture (mL) 3M Water NaOAc Ethanol Total 378.00027.000 40.327 445.327

The ethanolic Lipid Mixture was then added to the Aqueous Mixture whilerapidly stirring on a magnetic stir plate. Upon mixing, lipidoidvesicles formed spontaneously. The resulting vesicles were then extruded(2 passes) through a 0.08μ membrane (Whatman, Nucleopore) to size theempty vesicles. All manipulations were performed at room temperature.

Loading of Empty Vesicles with siRNA

An siRNA stock solution was prepared by dissolving desalted duplex siRNAin 50 mM sodium acetate pH 5 at a concentration of 10 mg/mL. Anappropriate volume of this siRNA stock was mixed with the appropriatevolume of ethanol to yield a diluted siRNA solution in 35% (vol) ethanol(see table below).

siRNA Dilution siRNA siRNA Stock (50 nM (mg/mL) NaOAc) Ethanol Total 10180.000 96.923 276.923

277 mL of diluted siRNA solution was added to 623 mL of empty vesiclemixture while rapidly stirring on a magnetic stir plate. The resultingcombined mixture was then incubated at 37° C. for 30 min to allow forloading of siRNA.

Ultrafiltration and Terminal 0.2 p Filtration

After incubation, the 900 mL loaded nanoparticle mixture was dilutedinto 1.8 L of PBS to yield a 2.7 L diluted mixture. This diluted mixturewas then concentrated to ˜1 L and diafiltered by tangential flowfiltration against 10 volumes of PBS using a Sartorius TFF systemutilizing two stacked 100,000 MWCO cartridges. No back-pressure wasapplied to the cartridge and the pump speed was set to 300 rpm. Afterbuffer exchange the resulting solution was concentrated to roughly 2mg/mL siRNA.

Terminal filtration was performed by passing the solution through a 0.2μfilter capsule (Whatman, Polycap 36 AS).

A flow chart illustrating this process is shown in FIG. 10.

Example 47 Comparison of Particle Size on Efficacy

Association complexes were formed using the procedure generallydescribed in Example 46. However, because the complexes were beingevaluated based on size, different extrusion membranes were used toproduce particles having the following diameters: 150 nm, 85 nm, 60 nm,and 50 nm. The siRNAs loaded in the complexes targeted factor VII.

The particles were evaluated in a Factor VII silencing assay,demonstrating that the 50 nm particles were the most efficaciousrelative to the 150 nm, 85 nm, and 60 nm particles. The results of theassay are depicted in FIG. 11.

Example 48 Comparison of Half Life of Nucleic Acid Agents UnformulatedVersus Formulated into an Association Complex

The half life of siRNA formulated in association complexes was evaluatedin vitro in human serum at 37° C. The association complexes wereprepared as in Example 46. For purposes of comparison, unformulatedsiRNA was also evaluated in vitro in human serum. The percent of fulllength product determined by HPLC was evaluated for both the formulatedand unformulated siRNA. As demonstrated in FIG. 12, the formulated siRNAhad a significantly improved half life in vitro in human serum.

Example 49 Comparison of Efficacy of Association Having Peg Lipids ofVaried Chain Length

Association complexes were prepared as in Example 46 with variation onthe length of the alkyl chain of the PEG lipid. Alkyl chain lengths of10, 11, 12, 13, 14, 15, and 16 were evaluated and compared for efficacyin a Factor VII silencing assay. As shown in FIG. 13, chain lengths of13, 14, and 15 demonstrated the most silencing as measured in the assay.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

What is claimed is:
 1. A compound having the formula (XV):

wherein L¹ and L² are both a bond; each R¹ and R² are independentlyalkyl, alkenyl, or alkynyl, each of which is optionally substituted withone or more substituents; X is —C(O)NH—, C(S)NH,—C(O)—C₁₋₃alkyl-C(O)NH—, or —C(O)—C₁₋₃alkyl-C(O)O—; m is an integer from0-11; and n is an integer from 1-500.
 2. The compound of claim 1,wherein R¹ and R² are independently unsubstituted C₆-C₂₈ alkyl.
 3. Thecompound of claim 1, wherein R¹ and R² are independently unsubstitutedC₁₀-C₁₈ alkyl.
 4. The compound of claim 1, wherein the substituents forR₁ and R² are selected from alkyl, cycloalkyl, haloalkyl, aryl,heteroaryl, arylalkyl, heteroarylalkyl, heterocyclyl, alkenyl, alkynyl,cycloalkenyl, heterocycloalkenyl, alkoxy, haloalkoxy, halo, hydroxy,carboxy, carboxylate, cyano, nitro, amino, alkyl amino, SO₃H, sulfate,phosphate, methylenedioxy (—O—CH₂—O— wherein oxygens are attached tosame carbon (geminal substitution) atoms), ethylenedioxy, oxo, thioxo,imino, S(O)_(n)alkyl (where n of S(O)_(n)alkyl is 0-2), S(O)_(n)aryl(where n of S(O),_(n)aryl is 0-2), S(O)_(n)heteroaryl (where n ofS(O)_(n)heteroaryl is 0-2), S(O)_(n)heterocyclyl (where n ofS(O)_(n)heterocyclyl is 0-2), amine, ester, amide, and sulfonamide.
 5. Acompound having the formula

wherein L¹ and L² are both a bond; each R¹ and R² are independentlyalkyl, alkenyl, or alkynyl; each of which is optionally substituted withone or more substituents; m is an integer from 0-11 and n is an integerfrom 1-500.
 6. The compound of claim 5, wherein the substituents for R¹and R² are selected from alkyl, cycloalkyl, haloalkyl, aryl, heteroaryl,arylalkyl, heteroarylalkyl, heterocyclyl, alkenyl, alkynyl,cycloalkenyl, heterocycloalkenyl, alkoxy, haloalkoxy, halo, hydroxy,carboxy, carboxylate, cyano, nitro, amino, alkyl amino, SO₃H, sulfate,phosphate, methylenedioxy (—O—CH₂—O— wherein oxygens are attached tosame carbon (geminal substitution) atoms), ethylenedioxy, oxo, thioxo,imino, S(O)_(n)alkyl (where n of S(O)_(n)alkyl is 0-2), S(O)_(n)aryl(where n of S(O)_(n)aryl is 0-2), S(O)_(n)heteroaryl (where n ofS(O)_(n)heteroaryl is 0-2), S(O)_(n)heterocyclyl (where n ofS(O)_(n)heterocyclyl is 0-2), amine, ester, amide, and sulfonamide. 7.The compound of claim 5, wherein R¹ and R² are independentlyunsubstituted C₆-C₂₈ alkyl.
 8. The compound of claim 5, wherein R¹ andR² are independently unsubstituted C₁₀-C₁₈ alkyl.
 9. The compound ofclaim 7, wherein R¹ and R² are straight chain alkyl having the samelength.
 10. The compound of claim 8, wherein R¹ and R² are straightchain alkyl having the same length.
 11. The compound of claim 5, whereinR¹ and R² are unsubstituted C₁₄ alkyl.
 12. The compound of claim 5,wherein R¹ and R² are unsubstituted C₁₈ alkyl.
 13. The compound of claim5, wherein m is
 2. 14. The compound of claim 5, wherein n is from 40 to400.
 15. The compound of claim 5, wherein n is from 40 to
 50. 16. Thecompound of claim 5, wherein n is from 42 to
 47. 17. The compound ofclaim 5, wherein L¹ and L² are both bonds, R¹ and R² are both alkyl, andn is an integer from about 40-400.
 18. The compound of claim 17, whereinR¹ and R² are unsubstituted C₆-C₂₈ alkyl.
 19. The compound of claim 17,wherein R¹ and R² are unsubstituted C₁₀-C₁₈ alkyl.
 20. The compound ofclaim 17, wherein R¹ and R² are unsubstituted C₁₄ alkyl.
 21. Thecompound of claim 5, wherein the compound has the formula:

wherein each R is C₁₄H₂₉ and n is from 42 to
 47. 22. The compound ofclaim 21, wherein the average molecular weight of the PEG moiety is2000.
 23. The compound of claim 5, wherein the compound has the formula:

wherein each R is C₁₈H₃₇ and n is from 42 to
 47. 24. The compound ofclaim 23, wherein the average molecular weight of the PEG moiety is2000.
 25. The compound of claim 5, wherein the compound has the formula:

wherein n is an integer from 1-500.
 26. The compound of claim 25,wherein n is between 42 and
 47. 27. A lipid particle comprising acompound of claim
 5. 28. The compound of claim 1, wherein, each of R¹and R² are independently C₆-C₂₈ alkyl or C₆-C₃₀ alkenyl and X is—C(O)NH—.